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The process of preparing small seed mixtures, especially of perennial cereal grasses, is intensified by certain difficulties. Research has shown that the process of separating mixtures can be enhanced by using a high-voltage electric field. As a result, the separation of quality seed and the seed without an embryo occurs.

A detailed analysis of technical means and methods of seeds separation, as well as the research results of domestic and foreign authors, have proved that one of the perspective methods for getting of high quality seed material is the pneumatic and electric separation.The analysis of research works and the modern technical level of pneumatic separators showed that insufficient attention was paid to the study of aerodynamic separation of small-seed crops, especially, cereal grasses. By the conditions of separation in a vertical pneumatic channel the cereal crops change the area of the midlength section as to the air flow forces, and the probability was increased of getting of seeds with germs to a biologically defective seed without germs. To increase the quality of pneumatic separation one can by the selective orientation of seeds in mixture with longer axis perpendicularly to the flow of air. It is possible to achieve these conditions by the additional force action on seeds (as particles). The response of this force action could be different for a healthy seeds (with germs) and for a non-viable (without embryo) or a weed seed. Such a force effect on the particles of small-seed crop mixtures can be realized by creating of a homogeneous electric field in a separation channel. This requires a theoretical and experimental study of force action on particles during the process of separation, also justification of parameters and operating modes of the pneumatic electric separator. This study is the preconditions for design improvement of the pneumatic electric separator.

The work is devoted to the study of seed separation process of perennial grasses on a pneumatic-electric separator. It highlights the main problems of their post-harvest treatment. The influence of the regulated parameters - the magnitude of the supply of seeds to the air-separator, the air flow rate and the electric field strength on the quality from the segregated seed material of the perennial rye-grass were determined by conducting a multifactorial experiment. The optimal values of the regulated parameters of the pneumatic electric separation are substantiated, in which the content of heavy separating admixture of weeds in the seed mixture of perennial rye-grass is the least. It is proved that the use in the technological line of seed preparation of perennial grasses of the pneumatic electric separator allows to ensure the quality of the seed material of the perennial rye-grass in accordance with the requirements of the existing standards.

The aerobic adaptation of anaerobically grown P. denitrificans carried out under conditions of limited growth is characterized by an exponential decrease of nitrite reductase activity and a sharp increase of cytochrome oxidase and a slow increase of NADH:cytochrome c oxidase reductase and succinate dehydrogenase activities. The adaptation in a minimal adaptation medium under conditions of active or blocked protein synthesis showed that in addition to the degradation component of turnover during the aerobic adaptation other degradation enzyme(s), whose synthesis is induced by oxygen, are involved. This degradation system plays an essential role in the rapid disappearance of nitrite reductase and a pronounced decrease of the membranebound cytochrome c oxidase activities during aerobic adaptation in the minimal adaptation medium. [ABSTRACT FROM AUTHOR]

Limited aeration of cell suspension in growth medium was used to study the kinetics of formation of nitrite reductase and nitrous-oxide reductase and their physiological electron donor, cytochrome c-550, during the anaerobic adaptation of Paracoccus denitrificans. The crucial step in the regulation of synthesis of these components is the repressive effect of oxygen while nitrogenous acceptors (NO , NO , NO) probably play no role as inducers. The time course of the enzyme activites was analogous (after a lag phase a sharp increase with a maximum after 3 h) and differed from the kinetics of synthesis of cytochrome c-550 (gradual rise throughout the 8-h experiment). [ABSTRACT FROM AUTHOR]

Concentration of cytochrome c decreases when shaking anaerobically grown cells in a non-growth medium down to 50% of the original amount in the cell, depending on the degree of aeration. Only 10-20% of this amount can be found in the adaptation medium. The main portion of cytochrome c is degraded during the adaptation. Inhibitors of proteinases do not influence the degradation. Addition of mammalian cytochrome c fully prevents the degradation of bacterial cytochrome c but not its release from cells. Potassium hexacyanoferrate(III) exhibits a similar effect as oxygen. The degradation system is probably localized in the periplasmic space of the cells. [ABSTRACT FROM AUTHOR]

The kinetics of the ubiquinol-cytochrome c reductase reaction was examined using membrane fragments and purified bc(1) complexes derived from a wild-type (WT) and a newly constructed mutant (MUT) strains of Paracoccus denitrificans. The cytochrome c(1) of the WT samples possessed an additional stretch of acidic amino acids, which was lacking in the mutant. The reaction was followed with positively charged mitochondrial and negatively charged bacterial cytochromes c, and specific activities, apparent k(cat) values, and first-order rate constant values were compared. These values were distinctly lower for the MUT fractions using mitochondrial cytochrome c but differed only slightly with the bacterial species. The MUT preparations were less sensitive to changes of ionic strength of the reaction media and showed pure first-order kinetics with both samples of cytochrome c. The reaction of the WT enzyme was first order only with bacterial cytochrome c but proceeded with a non-linear profile with mitochondrial cytochrome c. The analysis of the reaction pattern revealed a rapid onset of the reaction with a successively declining rate. Experiments performed in the absence of an electron donor indicated that electrostatic attraction could directly participate in cytochrome c reduction.

Cytochrome (cyt) c forms complexes, undergoes a conformational change and becomes partly reduced at interaction with membrane anchored alkaline phosphatase (AP), a glycoprotein which is released into the body fluid in forms differing in hydrophobicity. The proportion of products formed in the mixtures depends on pH, ionic strength, temperature and the buffer composition. The reaction terminates in an equilibrium between cyt c(FeII) and other cyt c conformers. Optimal conditions for the rate of the reaction are 100 mM glycine/NaOH, pH 9.7-9.9, at which 68-74% of cyt c is found in the reduced state. The interaction affects compactness of the haem cleft as shown by changes induced in CD spectra of the Soret region and changes in optical characteristics of phenylalanine, tyrosine and tryptophan residues. Differential scanning calorimetry of AP+cyt c mixtures revealed a creation of at least two types of complexes. A complex formed by non-coulombic binding prevails at substoichiometric AP/cyt c ratios, at higher ratios more electrostatic attraction is involved and at 1:1 molar ratio an apparent complexity of binding forces occurs. The rapid phase of the cyt c(FeII) formation depends on the presence of the hydrophobic alkylacylphosphoinositol (glycosylphosphatidylinositol) moiety, the protein part of the enzyme participates in an electrostatic and much slower phase of cyt c(FeII) creation. The results show that non-coulombic interaction may participate at interaction of cyt c with cellular proteins.

Two proteins, alkaline phosphatase (AP) and cytochrome c (cyt c) which seem to be involved in the apoptotic cell death program were examined on their interaction. Intestinal AP affects ferricytochrome c (cyt c(FeIII)) by changing its optical properties, redox state and conformation. The effect proceeded over the course of hours with a gradual decrease in free cyt c(FeIII) as the AP concentration increased. A heme containing high molecular species was created in the first stage of interaction of the proteins in neutral, acidic (pH 2.6), alkaline (pH 8.3), low ionic strength (10 mmol/l phosphate), and high ionic strength (0.5 mol/l NaCl) media. Further complexation was favored by higher pH values and temperature. Differential scanning calorimetry revealed a decrease in enthalpy of the thermodenaturation temperature (Tm) of cyt c at 84.5 degrees C due to the AP addition. Increments of AP in the mixtures resulted in the appearance of Tm peaks at 68 degrees C and 61 degrees C. Electrophoretic analysis of the commercial samples of intestinal APs showed main fractions from 63.2 kDa to 72.9 kDa and from 172.9 up to 179.0 kDa. Changes in positions and intensities of the bands were detected upon longer incubation (24 h) with cyt c. The electrophoretic pattern of the bacterial AP was homogeneous with one fraction of 43.7 kDa showing no alteration due to the cyt c presence. Gel permeation chromatography of incubated mixtures of intestinal APs and cyt c confirmed the creation of new heme containing complexes.

Alkaline phosphatase (AP) a protein which exhibits long-lived phosphorescence lifetime and ferricytochrome c as a phosphorescence quenching agent were examined. The excitation of the tryptophan triplet state resulted in cytochrome c reduction confirming long-range electron transfer as the quenching mechanism. The rate of electron transfer was not related to the length of the illumination interval; an additional reaction between the two proteins leading to cytochrome c reduction was detected. The reaction which proceeded in the dark was not sensitive to oxygen, was dependent on pH, and on the AP to cytochrome c ratio. At optimum 68 +/- 4% of the total cytochrome c could be reduced due to the presence of AP. On incubation of the two proteins the conformation of cytochrome c was altered as was evidenced by its decreased reducibility by ascorbate, by the disappearance of the absorption band at 695 nm, by the appearance of the new band at 620-640 nm, and by a change in circular dichroism spectra witnessing a structural alteration in the vicinity of the heme cleft. This was characterized by a profound increase in positive elipticity at 400 nm and by a reversible change in the magnitude of negative elipticity at 417 nm. The reaction was not significantly affected by the addition of sulfhydryl-binding and metal-complexing agents.

Cytochrome c oxidase binds protonophore carbonyl cyanide 3-chlorophenylhydrazone (CCCP) with high affinity. There are 1.46 high-affinity binding sites per cytochrome c oxidase for CCCP with dissociation constant 2.7 x 10(-7) mol/l. The bond between the CCCP and cytochrome c oxidase accomplishes through the group on cytochrome c oxidase with pKa 6.64 and is based on the electrostatic interaction. Interaction of CCCP with low-affinity binding sites of cytochrome c oxidase induces the shift of the anion CCCP spectrum to UV-region. The similar effect is characteristic for CCCP interaction with protons. Lipophilic non-dissociated derivative NCH3CCP is not binding to cytochrome c oxidase.

The rate of reduction of terminal acceptors (nitrate, nitrite, and oxygen) in anaerobically grown cells of Paracoccus denitrificans increased on permeabilization of cytoplasmic membrane. It was proved that under aerobic conditions the increase of the rate of nitrate reduction was caused by: (i) the abolishment of the permeability barrier for nitrate, (ii) the enhancement of the influx of redox equivalents to the respiratory chain due to the stimulation of succinate dehydrogenase reaction, and (iii) the inhibition of electron flow to oxygen by endogenously formed nitrite. Nitrite inhibits oxygen reduction by its interaction with the terminal part of the respiratory chain (I50 = 15 microM) localized at the inner aspect of the cytoplasmic membrane. The distribution of nitrite between intact cells and the suspension medium follows the Nernst equation for monovalent anion. The possible physiological consequences of the low intracellular nitrite concentration are discussed.

Pi uptake in cells or spheroplasts of Paracoccus denitrificans is biphasic; only the first rapid phase represents net Pi transport. The second phase is limited by the rate of Pi utilization inside the cell, i.e., mainly by its esterification, and as such it was inhibited by DCCD. The Pi/dicarboxylate antiporter does not seem to be operative, and its inhibitor n-butylmalonate did not exert specific inhibition. Pi transport is inhibited by SH reagents; the most potent inhibitor is PCMB, and mersalyl is much less effective. However, neither inhibitor affects efflux of accumulated Pi. The gradient of potassium ions may be involved in the Pi uptake, which is lowered in the presence of valinomycin. FCCP alone does not release accumulated Pi from spheroplasts unless they are preincubated with SCN-. The results indicate that Pi enters the cell by symport with protons.

We have established the participation of a mobile redox pool in the respiratory chain of anaerobically grown bacterium Paracoccus denitrificans. In testing the kinetical homogeneity of the pool it was found that the ratio of fluxes of electron transport toward the terminal acceptors oxygen and nitrate was coincident for the respiratory substrates NADH and succinate; this provides evidence against the preferential link of one dehydrogenase with a distinct terminal enzyme through the separate pool of ubiquinone. The deviation from the expected behavior observed in comparing the titration of NADH oxidase and succinate oxidase with respiratory inhibitors such as mucidin (inhibitor in the bc1 region) or cyanide can be accounted for by the activation of succinate dehydrogenase upon the increase in the reduced state of respiratory components during the titration.

The preferential utilization of oxygen, the terminal acceptor, in anaerobically grown cells of Paracoccus denitrificans was abolished in the presence of uncoupler (3 microM carbonyl cyanide m-chlorophenylhydrazone) which brought about a switch to the reduction of nitrite. It has been proved by measuring the redox state of cytochromes that this effect is due to the inhibition of the electron flow to oxygen caused by nitrite, which attains the site of its inhibitory action when the membrane potential is lowered.