Studies on the protolytic reactions coupled with water cleavage in photosystem II membrane fragments from spinach

The protolytic reactions of PSII membrane fragments were analyzed by measurements of absorption changes of the water soluble indicator dye bromocresol purple induced by a train of 10 s flashes in dark-adapted samples. It was found that: a) in the first flash a rapid H⁺-release takes place followed by a slower H⁺-uptake. The deprotonation is insensitive to DCMU but is completely eliminated by linolenic acid treatment of the samples; b) the extent of the H⁺-uptake in the first flash depends on the redox potential of the suspension. In this time domain no H⁺-uptake is observed in the subsequent flashes; c) the extent of the H⁺-release as a function of the flash number in the sequence exhibits a characteristic oscillation pattern. Multiphasic release kinetics are observed. The oscillation pattern can be satisfactorily described by a 1, 0, 1, 2 stoichiometry for the redox transitions S_i S_i+1 (i=0, 1, 2, 3) in the water oxidizing enzyme system Y. The H⁺-uptake after the first flash is assumed to be a consequence of the very fast reduction of oxidized Q₄₀₀(Fe³⁺) formed due to dark incubation with K₃[Fe(CN)₆]. The possible participation of component Z in the deprotonation reactions at the PSII donor side is discussed.Abbreviations A protonizable group at the PSII acceptor side - BCP Bromocresol Purple - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - FWHM Full Width at Half Maximum - Q_A, Q_B primary and secondary plastoquinone at PSII acceptor side - Q₄₀₀ redox group at PSII-acceptor side (high spin Fe²⁺) - P680 Photoactive chlorophyll of PSII reaction center - S_i redox states of the catalytic site of water oxidation - Z redox component connecting the catalytic site of water oxidation with the reaction center     

Reactive extraction of penicillin G in a pilot plant Karr-column

Summary Penicillin G was extracted from a model medium with a secondary amine (Amberlite LA-2) as carrier in n-butylacetate as solvent in a 7.6 m high pilot plant Karr-column at different stroke frequencies, throughput of the phases, concentrations of Penicillin G and carrier and ratios of the throughputs of the aqueous and organic phases. Up to penicillin concentrations of 30 gl^–1, throughputs of the aqueous phase of 100 lh^–1 and throughput ratios of the aqueous phase-to-organic phase of 3, very high degrees of extraction (99%) can be achieved with a penicillin loss below 1%.Symbols a specific interfacial area with regard to the volume of the continuous phase - C partition coefficient - cA, cA, i concentration of carrier (sec. amine) in the bulk at the interface - cAHP, cAHP, i concentration of complex in the bulk at the interface - cH proton concentration - cHP_a, cHP_a,i concentration of free acid in the bulk of the aqueous phase at the interface - cHP_o, cHP_{o, i} concentration of free acid in the bulk of the organic phase, at the interface - cP, cP, i concentration of acid anions in the bulk of the aqueous phase, at the interface - d₃₂ Sauter droplet diameter - E degree of extraction - f stroke frequency - KG reaction equilibrium constant - K_phys distribution coefficient - N number of stages in cascade - t mean residence time of the aqueous phase - _aq throughput of the aqueous phase - _o throughput of the organic phase - Z dimensionless longitudinal coordinate of the column with regard to its active length (4 m) - holdup of the organic phase     

Continuous acetone-butanol production with direct product removal

Summary To eliminate the product inhibition and increase the productivity of butanol formation, a continuously operated membrane bioreactor was connected to a four-stage mixer-settler cascade. Clostridium acetobutylicum was cultivated in this reactor. Butanol was selectively extracted with butyric acid saturated n-decanol from the cell-free cultivation medium, and the butanol-free medium was refed into the reactor. Due to the high boiling point of decanol, the recovery of butanol from the decanol solution is easy. The partition coefficient and selectivity of butanol in the cultivation medium-decanol-system is sufficiently high for removing it from the medium. Direct contact of the cells with the decanol phase causes cell damage. However, decanol is practically insoluble in the fermentation medium, thus the contact of the cell-free medium with the solvent phase does not influence of cell growth and product formation. At a dilution rate of D _z=0.1 h^-1, the butanol productivity was increased by removing butanol from the medium by a factor of four. A further increase was prevented by a contaminant of the technical decanol, which was identified by GC-MS-analysis as 1-,3-hexandiol.Symbols D dilution rate, h^-1 - D _eff effective dilution rate (Eq. 3), h^-1 - D _Ex extraction dilution rate (Eq. 3), h^-1 - D _g dilution rate of cell suspension in reactor-filter-system, h^-1 - E degree of extraction (Eq. 3), l - P product concentration in medium after extraction, g l^-1 - P _O product concentration in reactor, g l^-1 - R _P productivity and product formation rate, g l^-1 h^-1 - q _p S specific product formation coefficient with regard to the cell growth rate, l - V _F volume of cell suspension in filter module, l - V _g volume of the cell suspension in reactor and in filter module V _g =V _R +V _F , l - V _R volume of cell suspension in ractor, l - v _O cell free feed rate, l h^-1 - v ₁ flow rate of cell suspension leaves the reactor, l h^-1 - v _E flow rate of decanol through the extractor, l h^-1 - v _w flow rate of the cell free medium through the filter modul, l h^-1 - X cell mass concentration, g l^-1 - specific growth rate of the cells, h^-1 Dedicated to Professor Dr. H. J. Rehm on the occasion of his 60th birthday     

Reactive extraction of penicillin G in a pilot plant Karr-column II. Fermentation broths

Summary Penicillin G was extracted from mycelfree fermentation broths by means of the carrier (Amberlite LA-2) in n-butylacetate at pH 5 in a 7.6 m high pilot plant Karr-column with degrees of extraction E=98–99% and penicillin enrichments up to 3. The reextraction was carried out with phosphate buffer at pH-values above 7.5 with degree of extractions E=86–88% and penicillin enrichments up to 3. The penicillin and carrier losses were negligible. The influence of the process variables on the extraction degree was investigated. The penicillin extraction of the model medium and the fermentation broths were compared. Recommendations are given for the optimal penicillin recovery with reactive extraction.Symbols a specific interfacial area with regard to the volume of the continuous phase - cA concentration of carrier - cAHP,O concentration of complex in feed - cP,cP,O concentration of penicillin acid anion in theaqueous phase, in the feed - d ₃₂ Sauter droplet diameter - E degree of extraction - f stroke frequency - V _aq throughput of the aqueous phase - V ₀ throughput of the organic phase - Z dimensionsless longitudinal coordinate of the column with regard to its active length (4m) - holdup of the organic phase     

Prostanoid formation in primary astroglial cell cultures: Ca-dependency and stimulation by A 23187, melittin and phospholipases A2 and C

Prostaglandin (PG) and thromboxane B₂ (TXB₂) biosynthesis was studied in cultured astrocytes from neonatal rat brain hemispheres. After two weeks of cultivation, prostanoids were formed with the spectrum: PGD₂ > TXB₂ > PGF₂ > PGE₂, as measured by specific radioimmunoassays. Under basal conditions PGD₂ biosynthesis (9.55 ng/mg protein/15 min) was in the same order of magnitude as the sum of the other prostanoids. The formation of prostanoids was stimulated in a concentration dependent manner (up to 6–10 fold) by the calcium ionophore A 23187 (0.01–10 μM) as well as by melittin (0.01–5 μg/ml), phospholipase A₂ (10–40 U/ml) and phospholipase C (0.01–1 U/ml). Basal and evoked PG and TXB₂ biosynthesis depended on the availability of Ca²⁺, as demonstrated in Ca²⁺ free incubation medium containing Na₂EDTA (1 μM), or with verapamil (100 μM) and 3,4,5-trimethoxybenzoic acid-8-(diethylamino)-octylester-HCl (TMB-8, 1–100 μM). Indomethacin (10 μM), mepacrine (100 μM) and p-bromophenacylbromide (50 μ M) inhibited basal and evoked PG formation. Thin-layer chromatography (TLC) detection after incubation of the cells with [³H]arachidonic acid (1 μCi/ml, for 60 min) confirmed the results obtained by radioimmunoassay. Incubation of [³H]arachidonic acid labelled cells with inonophore or phospholipases, followed by lipid extraction and TLC, showed that A 23187 liberated [³H]arachidonic acid predominantly from phosphatidylethanolamine, whereas phospholipase A₂ and C reduced mainly the labelling of the phosphatidyl-inositol/-choline fraction. Potassium depolarization of the cells did not enhance prostanoid formation. Similarly, drugs with affinity to - or β-adrenoceptors, or to dopamine-, 5-hydroxytryptamine-, muscarine-, histamine-, glutamate-, aspartate-, GABA, adenosine- and opioid-receptors failed to stimulate prostanoid biosynthesis. Also compounds like angiotensin, bradykinin and thrombin were ineffective in this respect.

In conclusion, our results confirm that cultured astrocytes possess the complete pattern of enzymes necessary for prostanoid formation and hence might play a crucial role in brain prostanoid biosynthesis. Stimulation of prostanoid biosynthesis involves Ca²⁺-dependent activation of phospholipase A₂, cyclooxygenase reaction and further PG metabolism. However, the endogenous stimulus for enhanced prostanoid synthesis in the brain still has to be established.     

Intracellular localisation of phytochrome and ubiquitin in red-light-irradiated oat coleoptiles by electron microscopy

The intracellular localisation of phytochrome and ubiquitin in irradiated oat coleoptiles was analysed by electron microscopy. We applied indirect immunolabeling with polyclonal antibodies against phytochrome from etiolated oat seedlings or polyclonal antibodies against ubiquitin from rabbit reticulocytes, together with a goldcoupled second antibody, on serial ultrathin sections of resin-embedded material. Immediately after a 5-min pulse of red light-converting phytochrome from the red-absorbing (Pr) to the far-redabsorbing (Pfr) form-the label for phytochrome was found to be sequestered in electron-dense areas. For up to 2 h after irradiation, the size of these areas increased with increasing dark periods. The ubiquitin label was found in the same electrondense areas only after a dark period of 30 min. A 5 min pulse of far-red light, which reverts Pfr to Pr, given immediately after the red light did not cause the electron-dense structures to disappear; moreover, they contained the phytochrome label immediately after the far-red pulse. In contrast, after the reverting far-red light pulse, ubiquitin could only be visualised in the electron-dense areas after prolonged dark periods (i.e. 60 min). The relevance of these data to light-induced phytochrome pelletability and to the destruction of both Pr and Pfr is discussed.Abbreviations FR far-red light; Pfr - Pr far-red-absorbing and red-absorbing forms of phytochrome, respectively - R red light     

Crystallization and preliminary x-ray diffraction studies of recombinant human interleukin-1 beta

Recombinant human interleukin-1 beta has been crystallized into a tetragonal cell. The unit cell constants are a = b = 54.9 A, c = 76.8 A, and alpha = beta = gamma = 90 degrees. The crystals diffract to better than 1.9 A and are suitable for high resolution data collection. The crystallization conditions and general crystal data are presented.