Development of sugar beet leaves: contents of hormones, localization of abscisic acid, and the level of products of photosynthesis.

The level of hormones in the tissues of sugar beet leaves of different age in parallel with their growth and metabolic activity was assayed; the latter was analyzed, measuring the contents of sugars and N-containing compounds, and the activities of Rubisco and proteases. The highest auxin and ABA concentration was detected in the actively growing upper leaf, while high level of cytokinins was maintained in the middle and upper leaves characterized by intensive photosynthesis. Leaf senescence being manifested in decline of chlorophyll content, decrease of photosynthesis and activation of proteolysis was accompanied by a decline in concentration of cytokinins. Glucose level gradually increased from upper (younger) to a lower (elder) leaves; this was accompanied with the signs of senescence on the background of decreased cytokinins level. Immuno-histochemical technique revealed increased level of abscisic acid in phloem parenchyma of the lowest leaf. The results suggest a possible involvement of auxins in maintaining leaf growth, an implication of decreased cytokinins level in the hypothesized induction of senescence by glucose, and a participation of abscisic acid in the active loading of metabolites into the phloem of senescing leaf.

[The "crystals" in the sugar beet (Beta vulgaris L.) leaves].

Crystal containing cells widely distributed in plant tissues, though the origin of the crystals and their functions are still opened to question. Membrane vesicles in beet leaves are visible in electronic microscope. They originate in cytoplasm and penetrate into vacuole by pinocytosis with participation of tonoplast. In light microscope, vesicles are luminous likewise crystals in crystal cells. Such vesicles-"crystals" fulfill crystal cells also. The content of vesicles-"crystals" are electronic transparent at every path of leaf development. It was proposed that distinct vesicles-"crystals" in cytoplasm and vacuole and mass of them in crystal cells, vein bundles, and epidermal cells--all of them are lytic compartments. Later, obviously, true crystals are formed.

Activity and carboxylation specificity factor of mutant ribulose 1,5-bisphosphate carboxylase/oxygenase from Anacystis nidulans.

The values of molecular carboxylase activity kcat and carboxylation specificity factor tau for mutant ribulose 1,5-bisphosphate carboxylase (rubisco) from Anacystis nidulans decreased as compared to those of the wild type recombinant rubisco. The substitution of five amino acid residues in rubisco large subunit Lys,Ala,Ser,Thr,Leu(339-343)Phe,Leu,Met,Ile,Lys had kcat decreased by 90% and tau by 36.3%. The same parameters for mutants with the single replacements decreased: for Thr342Ile kcat by 40.5% and tau by 16.7%, and for mutant Leu343Lys kcat by 48.1% and tau by 18.5%. Mutant rubisco with three amino acid residues changed Val,Asp,Leu(346-348)Tyr,His,Thr was inactive. The substitution Leu326Ile decreased kcat by 54.4% and tau by 34.2%; and change Ser328Ala decreased kcat only by 5.6% but tau by 41.5%. Replacement Asn123His decreased kcat by 16.5%. Significance of the non conservative amino acid residues for carboxylase activity and ribulose-1,5-bisphosphate partition is discussed.

[Characteristics of the autoregulatory factor d2 causing autolysis of Pseudomonas carboxydoflava and Bacillus cereus cells]. / Kharakteristika autoreguliatornogo faktora d2, vyzyvaiushchego avtoliz kletok Pseudomonas carboxydoflava i Bacillus cereus.

In the course of organoheterotrophous growth, Pseudomonas carboxydoflava Z-1107 and Bacillus cereus 504 were found to synthesize the autoregulatory factor d2, a membranotropic lipid metabolite, and to accumulate it in the growth medium. At a low concentration, the factor activated respiration of the cells: at a high concentration, it inhibited respiration and induced autolysis of the cells. The physiological action of d2 was shown to be due to the effect of free fatty acids, in particular, palmitic, stearic and oleic acids. Oleic acid displayed the highest physiological activity, P. carboxydoflava Z-1107 became more susceptible to high concentrations of d2 and oleic acid, and respiration of the cells was activated by low concentration of these compounds when the culture passed from the exponential growth phase to the linear one. These results as well as data reported in literature about the action of fatty acids on biological membrane suggest that low concentrations of the factor d2 uncouple respiration and oxidative phosphorylation whereas high concentrations of the factor disorganize the structure of the cytoplasmic membrane by increasing its fluidity.

[Organotrophic growth and the synthesis of an autoregulatory factor in Pseudomonas carboxydoflava]. / Organotrofnyi rost i sintez autoreguliatornogo faktora u Pseudomonas carboxydoflava.

The organotrophic growth of Pseudomonas carboxydoflava Z-1107 was studied in media limited or non-limited with respect to carbon and nitrogen sources. The organotrophic growth was found to decelerate not only when carbon and nitrogen sources were exhausted but also when their content was high enough. At all of the growth phases, P. carboxydoflava could synthesize butanol-soluble substances capable of inhibiting the respiration and growth of the microorganism (the autoregulator factor d) and release them into the cultural broth. The dynamics of accumulation of the factor d in the cultural broth was determined using an arbitrary criterion (the inhibition of respiration in the control culture). The authors discuss a possibility to explain the deceleration of P. carboxydoflava organotrophic growth in a medium which is not limited with carbon and nitrogen sources in terms of the accumulation of the factor d in the culture, as well as possible reasons for the absence of a direct correlation between the activity of the factor and the growth rate of the culture.

[Carboxysomes of the thermophilic hydrogen bacterium Pseudomonas thermophila]. / Karboksisomy termofil'noi vodorodnoi bakterii Pseudomonas thermophila.

The technique of electron microscopy was used to detect the presence of paracrystal hexagonal inclusions in the cells and spheroplasts of the thermophilic hydrogen bacterium Pseudomonas thermophila. The connection of the inclusions with DNA threads can easily be seen on photomicrographs of the spheroplasts. The cells were disintegrated by freezing and thawing and the resultant homogenates were centrifuged in a sucrose density gradient; up to 80% of the activity of ribulose-1,5-diphosphate carboxylase (RDP carboxylase, EC 4.1.1.39) was found in the fraction of particles containing hexagonal paracrystal inclusions (carboxysomes). Their granular content and the tendency to diffuse were seen at a magnification x100,000. The percentage of the insoluble enzyme was higher in cells in the stationary growth phase than in growing cells. Only 25% of the enzyme activity was detected in the particles after the cells treated with lysozyme had been subjected to osmotic shock. A possible role of carboxysomes in cells as a compartment storing RDP carboxylase is discussed.

[Ultrastructural organizational characteristics of the hydrogen thermophilic bacterium, Pseudomonas thermophila]. / Osobennosti ul'trastrukturnoi organizatsii vodorodnoi termofil'noi bakterii Pseudomonas thermophila.

The ultrastructural organization of the lithotrophic hydrogen bacterium Pseudomonas thermophila K2 was described for the first time and was found to be typical of Gram-negative bacteria. The ultrastructure of this organism is characterized by (i) irregular plication of the outer membrane of the cell wall and a very thin (2-3 nm) rigid layer; (ii) a considerable number of intracellular membranes differing in their structure and location; (iii) fragmentation of the cytoplasm involving the plasmalemma and the cell wall; (iv) the presence, in the nucleoid zone, of paracrystals having a hexagonal shape and resembling carboxysomes in their size, shape and macromolecular organization, which had not been found in hydrogen bacteria hitherto.

[Carbon dioxide fixation and its regulation in Synechococcus (Anacystis nidulans) cyanobacteria]. / Fiksatsiia uglekisloty i ee reguliatsiia u tsianobakterii Synechococcus (Anacystis nidulans).

The bulk of 14CO2 assimilated in photosynthesis by the growing culture of Synechococcus was incorporated in the cell via the reductive pentose phosphate cycle. Up to 70% of the label was incorporated into phosphoglyceric acid and phosphoric esters of sugars at all stages of the active cultural growth after 1 min of exposition in the light in the presence of NaH14CO3. The relative proportion of the label in phosphorylated compounds of the reductive pentose phosphate cycle decreased if the exposition was increased to 20 min. The content of 14C in aspartic acid did not exceed 9%. In the presence of nucleotide peptide (NP) isolated from Anabaena variabilis, the overall rate of carbon dioxide assimilation rised by 50% as compared to the control by the fourth day of the growth. The specific rate of 14CO2 assimilation hardly changed within four days of the cultural growth; it was 44 nmole/min/mg, or 55 nmole/min/mg in the presence of NP. NP had no effect on the qualitative composition of the products of photosynthesis; however, the percentage of sugar phosphates, phosphoenolpyruvate and organic acids (carbohydrates at the stage of exponential growth) in these products increased in the presence of NP (exposition for 20 min). The percentage of carbohydrates was found to change only slightly as compared to the control in the presence of NP. The content of protein and RNA increased by 25-30% and the content of DNA by 60%. The action of NP was most pronounced if the content of DNA was calculated per cell. Therefore, there is a correlation between the content of DNA and the intensification of carbon dioxide fixation by the culture.

[Functional characteristics of the membrane preparations from the cells of the thermophilic hydrogen bacterium. Pseudomonas thermophila]. / Funktsional'naia kharakteristika membrannykh preparatov iz kletok termofil'noi vodorodnoi bakterii Pseudomonas thermophila.

The electron transport chain (ETC) of Pseudomonas thermophila K-2 was examined by the amperometric determination of O2 uptake by the preparations of membranes isolated by ultracentrifugation at 14,000 g. Amytal and cyanide were found to inhibit endogenous respiration of membranes from freshly grown cells. The membrane preparations, after exhaustion of endogenous substrates in them, oxidized NADH and succinate at rates of 4.00 and 0.83 mumole/min per 1 mg of membrane protein, respectively. The oxidation of NADH was inhibited by rotenone and cyanide, while the oxidation of succinate, only by cyanide. Maxima corresponding to NADH and iron-containing proteins were found in the fluorescence spectra of membrane preparations from Ps. thermophila K-2. Endogenous NADH was not susceptible either to incubation of the preparations in the air, or to hydrogen being bubbled through. These results and the data reported in literature make it possible to conclude that the membrane preparations from Ps. thermophila K-2 contain all the ETC components similar to the mitochondrial ones.

[Effect of temperature and pH on the ribulose-1,5-diphosphate carboxylase activity of the thermophilic hydrogen bacterium, Pseudomonas thermophila]. / Vliianie temperatury i pH na aktivnost' ribulozo-1,5-difosfatkarboksilazy termofil'noi vodorodnoi bakterii Pseudomonas thermophila.

The activity of ribulose 1,5-diphosphate (RDP) carboxylase was found in the soluble fraction of the cytoplasm from sonicated Pseudomonas thermophila K-2 cells. The enzyme is relatively thermolabile and completely loses its activity at 80 degrees C. The activity of RDP carboxylase at 60 degrees C increases by 40% during the first 10 min of heating in the presence of Mg2+ ions, bicarbonate and dithiothreitol, and again decreases if the enzyme is heated over 20 min. The optimum temperature of the enzyme is 50--55 degrees C. The specific activity of the enzyme in fresh preparations under these conditions reaches 0.22 unit per 1 mg of protein in the extract. The calculated value of the activation energy for RDP carboxylase is 6.4 kcal-mole-1, but 11.6 kcal-mole-1 in frozen preparations. The optimal pH is 7.0--7.3 depending on the buffer. The temperature optimum for the enzyme action does not depend on pH within the range of 7.3 to 8.8. Therefore, RDP carboxylase of Ps. thermophila K-2 differs from RDP carboxylases of mesophilic cultures studied earlier by a higher susceptibility to a decrease in temperature (the enzyme activity is negligible at 30 degrees C), by a lower value of the activation energy at suboptimal temperatures, and by a lower pH optimum of the enzyme action.

[Localization of hydrogenase in the cells of the thermophilic hydrogen bacterium, Pseudomonas thermophila]. / Lokalizatsiia gidrogenazy v kletkah termofil'noi vodorodnoi bakterii Pseudomonas thermophila.

Spheroplasts that were osmotically stable in 0.2M Tris-HCl--0.02M EDTA were prepared from the autotrophically grown cells of Pseudomonas thermophila K-2. The spheroplasts possessed 90--95% of the hydrogenase activity of the whole cells. The half-life time of hydrogenase in the spheroplasts at 80 degrees C was 8.5 min. A spectrophotometric technique was developed for determining the membrane-bound hydrogenase in the presence of sulfhydryl compounds with methylene blue as electron acceptor. The maximal specific activity of hydrogenase in extracts prepared in the anaerobic conditions in the presence of dithiothreitol and Mg2+ and Mn2+ ions was 10 +/- 3 units per 1 mg of protein, which closely corresponded with the activity of hydrogenase in the whole cells. Almost all activity of hydrogenase assayed with methylene blue was localized in the membrane fraction. The activity of soluble NAD-specific hydrogenase was not detected. Large particles located in 60-70% sucrose had the highest hydrogenase activity upon fractionation in a continuous sucrose concentration gradient. The second, lower peak of the hydrogenase activity was detected in fractions of 40--50% sucrose. As was found by electron microscopy, the size of membrane vesicles with the hydrogenase activity varied within the range of 68--156 nm. The membrane preparations possessed the activity of NADH-dehydrogenase, NADH-oxidase and succinate dehydrogenase as well.

[Carbon assimilation pathways in the methylotrophy of Pseudomonas gazotropha]. / Puti assimiliatsii ugleroda pri metilotrofii Pseudomonas gazotropha.

The dynamics of label distribution was studied in the products of 14CH3OH assimilation by the cells of Pseudomonas gazotropha Z-1156. Substances to be first detected were glycolate, glycine and those of the chromatogram "start" spot. Later, the radioactivity was detected in phosphorylated compounds and glycerate. Cell extracts of Ps. gazotropha Z-1156 contained ribosephosphate isomerase, phosphoribulokinase and glyceraldehyde dehydrogenase but not ribulosediphosphate carboxylase. Distribution of the label in the products of 14CH3OH assimilation and the presence of active hydroxypyruvate reductase in the extract suggest that the serine cycle is involved in methylotrophy of Ps. gazotropha Z-1156. This suggestion is confirmed by the presence of active formate dehydrogenase, phosphoenolpyruvate carboxylase, (NADP+, Mn2+)-specific isocitrate dehydrogenase, (NAD, Mg2+)-specific malate dehydrogenase, malate lyase, and isocitrate lyase. The citric acid cycle is open at the alpha-ketoglutarate dehydrogenase system. The dry biomass of Ps. gazotropha Z-1156 contains over 70% of protein.

[Biochemical pathways of carbon monoxide assimilation in the carboxydobacterium, Pseudomonas gazotropha]. / Biokhimicheskie puti assimiliatsii ugarnogo gaza u karboksidobakterii Pseudomonas gazotrophe.

The incorporation of 14CO into acid-stable assimilation products by Pseudomonas gazotrophia Z-1156 is characterized by a slow rate at the beginning, contrary to the rectilinear kinetics for incorporation of the bicarbonate 14C in the presence of 12CO. The assimilation of 14C-bicarbonate decelerates in the absence of CO. The relative content of 14C is the highest in phosphorylated compounds upon the shortest possible incubation of the cells of P. gazotropha Z-1156 (5 min) in the presence of 14CO and O2, and decreases in the process of incubation. The bulk of radioactivity is found in aspartate and glutamate. The composition of products formed upon the assimilation of 14CO and NaH14CO3 (in the presence of 12CO) during 15 min is similar. The key enzymes of the reductive pentose phosphate cycle have been found in the cell extracts of P. gazotropha A-1156. The specific activity of carboxylating enzymes of the Calvin cycle in the cell extracts increases in the course of proportional growth and sharply decreases when the growth of the culture decelerates. The activity of ribulose diphosphate carboxylase (EC. 4.1.1.39) is always by one-two orders lower than that of ribulose phosphate isomerase (EC. 5.3.1.6) and phosphoribulokinase (EC.2.7.1.19), but is similar to the activity of phosphoenolpyruvate carboxylase (EC. 4.1.1.31). The activity of phosphoenolpyruvate carboxykinase (EC. 4.1.1.32) has not been detected in the cells extracts of P. gazotropha Z-1156.

[Phosphoenolpyruvate carboxylase in the carboyxdobacterium, Pseudomonas gazotropha]. / Fosfoenolpiruvatkarboksilaza karboksidobakterii Pseudomonas gazotropha.

The activity of phosphoenolpyruvate carboxylase (orthophosphate: oxalacetate-carboxy-lyase phosphorylating, E. C. 4.1.1.31) in the cell extracts of the carboxydobacterium Pseudomonas gazotropha Z-1156 depends on the presence of bivalent metal ions, Mn2+ ions being more effective than Mg2+ ions. The value of apparent KM for phosphoenolpyruvate in a freshly prepared extract is 7.1 mM. The affinity of the enzyme to phosphoenolpyruvate increases after storage of the extract in ice in the presence of dithiothreitol: KM=0.42 mM at low concentrations of the substrate, and 2.5 mm, at high concentrations of the substrate. The calculated maximum rate is 18.1 mE per 1 mg of protein of the extract, and changes only slightly upon storage in the presence of a stabilizer of sulphydryl groups. The activity of the enzyme reaches its maximum at the phase of deceleration of growth. Nucleotide triphosphates inhibit the activity of the enzyme more than the corresponding nucleotide diphosphates. The properties of PEP-carboxylase are discussed from the viewpoint of comparative biochemistry.

[Pathways of assimilation of carbon oxides in carboxydobacteria Seliberia carboxydohydrogena and Achromobacter carboxydus]. / Puti assimiliatsii okislov ugleroda u karboksidovakterii Seliberia carboxydohydrogena i Achromobacter carboxydus.

Assimilation products of 14C-bicarbonate and carbon-14C oxide were studied in two carboxydobacteria Seliberia carboxydohydrogena and Achromobacter carboxydus which differed in their ability for chemolithoautrophous growth in the presence of hydrogen. The dynamics and composition of labeled products formed upon assimilation of 14C-bicarbonate in the presence of unlabeled carbon oxide by the two organisms, the composition of products formed upon assimilation of 14CO by suspensions of S. carboxydohydrogena Z-1062 during 5 minutes, and the dynamics and composition of labeled assimilates of A. carboxydus Z-1171 after incubation in the presence of 14CO, were found to be consistent with those expected in the action of the reductive pentose phosphate Calvin cycle. The similarity of products formed upon assimilation of 14CO2 and 14CO suggests that CO is first oxidized to CO2, and only is assimilated.

[Hydrogenase activity of the thermophilic hydrogen-oxidizing bacterium Pseudomonas thermophila]. / Gidrogenaznaia aktivnost' termofil'noi vodorodokisliaiushchei bakterii pseudomonas thermophila.

The hydrogenase activity of the intact cells of a thermophilic hydrogen-oxidizing bacterium Pseudomonas thermophila K-2 was determined using methylene blue; it was several times higher than the rate of hydrogen uptake in the presence of oxygene and carbon dioxide. The activity of membrane-associated hydrogenase was assayed with the aid of phenazine methosulphate and 2,6-dichlorphenolindophenol as a cascade electron carrier. The enzyme is sufficiently stable in the air. The stability increases in the atmosphere of hydrogen. The membrane-bound enzyme was activated by Mn2+ ions. The pH-optimum of the enzyme activity in 0.1 M Tris-HCI buffer was 8,5-9,0. Natural electron acceptors tested, such as NAD, FMN, riboflavin, and cytochrome c, had no effect on the reaction rate. The enzyme is relatively thermostable: its activity was halved after heating at 78 degrees C for 10 min or at 80 degrees C for 8 min. Energy of activation was calculated. It was 14.5 kcal-mol-1 within the range of 23-40 degrees C and 10.3 kcal-mol-1 within the range of 40-60 degrees C.

[Assimilation of carbon dioxide by Stibiobacter senarmontii]. / Assimiliatsiia uglekisloty kul'turoi Stibiobacter senarmontii

Early products of 14CO2 assimilation by a new microorganism Stibiobacter senarmontii are phosphoglyceric acid, phosphorous esters of sugars and aspartic acid, as was shown by chromatography and radioautography. Extracts of the cells displayed the activity of ribulosediphosphate carboxylase and phosphoenolpyruvate carboxylase (1 mU and 0.24 mU per 1 mg of protein in the extract, respectively). Therefore, the microorganism is capable of autotrophic nutrition involving mechanisms of the reductive pentosephosphate cycle. The latter seems to operate even in conditions of deficiency of the energy substrate which is caused by low solubility of antimony trioxide.