Band 40, Heft 1-2

3,4-Diacetoxybutinylbithiophene was found to occur in considerable amounts in roots and hypocotyls of young Tagetes seedlings. This compound was converted enzymatically to 3,4-di- hydroxybutinylbithiophene by crude extracts from this plant. Experiments performed with enzyme preparations from plants of different stages of development supported the existence of two position-specific, subsequently working dithiophene acetate esterases. It is suggested that 3-hydroxy-4-acetoxybutinylbithiophene is the intermediate product, formed by an highly specific 3,4-diacetoxybutinylbithiophene: 4-acetate esterase.

Two isomeric C-glycosides of apigenin (apigenin-6-C-glucosyl-8-C-arabinoside (schaftoside), apigenin-6-C-arabinosyl-8-C-glucoside (isoschaftoside)) were isolated from leaves of Ficus carica with preparative HPLC. 1The glycosides were identified by UV-, 1 H-NMR-, 13 C-NMR-spectroscopy and FAB-MS. Their concentration in fruits and leaves were determined by gradient HPLC on reversed phase material.

Dehydroabietane and an other diterpene hydrocarbon, being still unidentified, were found in the steam distillates of callus cultures of Melissa officinalis. The relative proportions of these two diterpenes changed considerably during the course of a cultivation passage. With advancing age of the cells they shifted gradually towards dehydroabietane. Cultures being exposed to conti­nuous light showed the same composition of their steam distillates as cultures which were raised in the dark.

A cell suspension culture of Catharanthus roseus was treated during the growth phase with the nucleoside analogue 5-azacytidine, a powerful inducer of cell differentiation, as evidenced with animal and human cell lines. After induction the cell culture was further incubated in a production medium in the absence of the inducer for 10 days. The extraction of the freeze dried cells and analysis by high performance liquid chromatography showed an additional intense peak in the elution profile, not present in extracts of untreated cells. The structure of the newly synthesized metabolite, elucidated by mass spectrometry and 1 H and 13 C NMR, was a lignan type compound, lirioresinol B mono-β-D-glucoside. This compound has hitherto not been found in Catharanthus roseus plants or cell cultures.

The response of yeast cells to different kinds of “stress” is not identical. Cells of the stationary growth phase synthesize three new proteins of molecular weights 68, 27 and 24 kD, compared with cells of the exponential growth phase, while heat-shocked cells exhibit new proteins of 100, 90. 84, 70 and 24 kD. After treatment with acrylonitrile two new proteins with molecular weights of 70 and 46 kD appear. However, all three kinds of “stress” lead to the induction of a ribonuclease.

When γ-glutamyltranspeptidase activity in tobacco cells was measured using the artificial substrate γ-glutamyl-p-nitroanilide, liberation of p-nitroaniline was not reduced, but stimulated by addition of glutathione. Therefore, glutathione was not acting as a donator, but as an acceptor of γ-glutamyl moieties in the assay mixture, suggesting that γ-glutamvltranspeptidase is not participating in degradation of glutathione. Feeding experiments with [ 35 S-cys]glutathione sup­ported this conclusion. When tobacco cells were supplied with this peptide as sole sulfur source, glutathione and γ-glutamylcysteine were the only labelled compounds found inside the cells. The low rate of uptake of glutathione apparently prevented the accumulation of measurable amounts of radioactivity in the cysteine pool. A γ-glutamylcyclotransferase, responsible for the conversion of γ-glutamylcysteine to 5-oxo-proline and cysteine was found in ammonium sulfate precipitates of tobacco cell homogenates. The enzyme showed high activities with γ-glutamylmethionine and γ-glutamylcysteine, but not with other γ-glutamyldipeptides or glutathione. From these and previously published experiments [(Rennenberg et a!., Z. Naturforsch. 35c, 708-711 (1980)], it is concluded that glutathione is degraded in tobacco cells via the following pathway: γ-glu-cys-gly → γ-glu-cys → 5-oxo-proline → glu.

Ion exchange and affinity chromatography techniques, similar to those previously reported for purification of adenosine kinase from human placenta, were applied to purification of rat liver adenosine kinase. The enzyme, purified 400-fold in 41% yield, was homogeneous on SDS- polyacrylamide gel electrophoresis, with a molecular weight of 52000. It specific activity, 18 μmol/min/mg protein, is the highest hitherto reported for this enzyme from mammalian sources. Chromatography on DEAE-cellulose removed about 98% of the phosphorylating activity towards 2′-deoxyadenosine present in the initial pH-treated liver extract. The final preparation exhibited only minimal activity (~ 1.5%) under optimal conditions (pH 7.5) vs- 2′-deoxy- adenosine, the lowest yet reported for such a preparation, with a K m of 670 μᴍ, as compared to 0.3 μᴍ for adenosine. The residual activity towards deoxyadenosine is considered an intrinsic property of the purified adenosine kinase and, in fact, phosphorylation of adenosine was inhibited competitively by deoxyadenosine, with a of 70 μᴍ. Competitive inhibition was also exhibited by cordycepin (3′-deoxyadenosine) with a K i of 150 μᴍ. A more potent competitive inhibitor was tubercidin, the K i for which was 1.9 μᴍ.

Hypertrehalosaemic hormones I and II from the corpus cardiacum of the American cockroach (Periplaneta americana) were separated by reversed-phase high-performance liquid chromato­graphy using a Nucleosil C 18 column with a trifluoroacetic acid/acetonitrile gradient. The eluent was monitored at 206 nm and the hypertrehalosaemic activity detected by bioassay. The amino acid compositions of hypertrehalosaemic hormone I and II were determined after acid hydrolysis with HCl or methanesulfonic acid. Both neurohormones are octapeptides. Hypertrehalosaemic hormone I contained the amino acids Asp (2) , Ser, Glu, Pro, Val, Phe and Trp, whereas hyper­trehalosaemic hormone II contained the amino acid residues Asp, Thr (2) , Glu, Pro, Leu, Phe and Trp.

The scent secretions of two subspecies of Hebomoia glaucippe, which are pierid butterflies, and the essential oils of their food plant were investigated by means of GC and GC-MS. The scent substances of either subspecies are found to be only E-β-ocimene, and the volatile components of Crataeva religiosa were identified as 2-hexanal, 3-hexen-1-ol, p-cymene, limonene, linalool, α-ionone, and β-ionone. Electroantennogram (EAG) were recorded with the antennae of adult male and female butterflies. It was found that EAG responses of male to E-β-ocimene were greater than those of females.

Four extracellular glycolipids produced under growth-limiting conditions were isolated from the culture broth of Pseudomonas spec. DSM 2874. After purification by column and thick-layer chromatography they were identified as anionic rhamnolipids. 1 H and 13 C-NMR studies showed that two of these, β(β(2-O-α-ʟ-rhamnopyranosyloxy)decanoyl)decanoic acid and β(β(2-O-α-ʟ- rhamnopyranosyl-α-ʟ-rhamnopyranosyloxy)decanoyloxy)decanoic acid, were identical with com­pounds described previously, while the other more hydrophilic compounds, β(2-O-α-ʟ-rhamno- pyranosyloxy)decanoic acid and β(2-O-β-ʟ-rhamnopyranosyl-a-ʟ-rhamnopyranosyloxy)decanoic acid, were new compounds. Surface and interfacial activity of the organic crude extract and of the purified components were determined in different aqueous solutions. The pH-dependence of surface and interfacial properties of the two previously described rhamnolipids (4. 20. 23) were examined in Teorell- Stenhagen-buffer (supplemented with 10% NaCl) at pH 3.0 and pH 9.0. All rhamnolipids reduced the surface-tension from 72 to about 30 mɴ/m and the interfacial-tension from 42 to about 1 mɴ/m. The critical micelle concentrations were of the order of 5 to 200 mg/l depending on the structure of the molecule.

In a simple phosphate buffer or a sodium chloride solution resting cells of Pseudomonas spec. DSM 2874 produced up to 15 g/l of different rhamnolipids. The rhamnolipid composition of the organic crude extract depended on the temperature during the cultivation and on the C-source. The optimal sodium chloride concentration for rhamnolipid formation was about 100 mᴍ/l and the optimal phosphate buffer concentration about 65 mᴍ/l. The optimal pH-value for the production of rhamnolipids from n-alkanes or glycerol was in the range pH 6.0-7.2. While rhamnolipid formation with glycerol as the sole C-source showed a wide optimum ranging from 27° up to 37 °C, production of rhamnolipids from n-alkanes had a sharp optimum at 37 °C. The addition of multivalent cations, different N-sources and EDTA caused an inhibition of rhamnolipid formation, while the n-alkane concentration had no influence. Specific rhamnolipid formation decreased with increasing cell concentration. Various C-sources were suitable for the formation of rhamnolipids by resting cells of Pseudomonas spec. DSM 2874. Yields, which were comparable to those obtained on n-alkanes or glycerol, were found for stearic acid, fatty alcohols and vegetable oils. A study of the time course of glycolipid production of resting cells was carried out in a 20 1- biorcactor with an intcnsor system and with n-tetradecane as the sole C-source.

The phospholipase C-activity in crude extracts of bovine blood platelets is strongly inhibited by the calmodulin-inhibitors fluphenazine and calmidazolium in the mᴍ range, and activated by ATP and ADP. but not by AMP. The activating effect is also shown by the nonhydrolysable ATP- and ADP-analogs α,β- and β,γ-methyleneadenosine 5′-triphosphate and α,β-methylene- adenosine 5′-diphosphate, thus indicating that it is an allosteric effect. The stimulation of the phospholipase C-activity by ATP is also detectable in some partially purified fractions of the crude platelet extract, but it is abolished on further purification of the enzyme.

Organelle membranes subfractionated from nodules of Glycine max differed in lipid compo­sition. Of the enzymes assembling phospholipids, choline kinase (EC 2.7.1.32) was recovered in the cytoplasmic fraction whereas CDP choline 1,2-diacylglyceride choline phosphotransferase (EC2.7.8.2) was located in the ER. When methyl [ 14 C]CDP choline was supplied to nodules in vivo radioactivity was found in the ER. On longer labelling periods [ 14 C]phosphatidylcholine spread into denser regions of the gradient, including the peribacteroid membrane (which surrounds the Rhizobium japonicum symbiont). [ 14 C]Phosphatidylcholine was a notable component of such membranes. Adenosine triphosphatase (EC 3.6.1.3) activity in the peribacteroid membrane fraction was compared to that of other major organelle fractions. The peribacteroid membrane ATPase was equally active on pH 6.0 and 8.0, was stimulated by K + and inhibited by DES, DCCD and MoO 4 . In these respects it was more similar to the ATPase activity in the ER and mitochondria than in tonoplast or cytoplasm. The results are discussed in relation to modes of peribacteroid membrane biogenesis, in­cluding enzyme modification.

The influence of 5-heptadecenylresorcinol and total rye 5-alkenylresorcinols isolated from rye grains on the red blood cell water permeability was studied using osmotic shrinkage experiments performed in 300 mᴍ sucrose. The studied compounds induced significant increase of erythro­cyte water permeability. The threshold concentration needed for the increase of water per­meability was in an order of 10 -6 mol/l. The temperature dependence of the observed process showed the discontinuity which was related to the 5-alkenylresorcinol transition temperatures. It was shown also that alkenylresorcinols did not exert the biphasic action on hypotonic lysis of erythrocytes usually observed for water soluble surfactants. The specific lysine activity is postulated for the studied compounds.

The effects of glucose and sucrose on the ionic conductance properties of the channel formed by Megathura crenulata hemocyanin in planar lipid bilayers have been studied using membranes of different compositions. It was found that glucose at high concentrations strongly affects the time constants of the current relaxations observed in membranes containing many channels after a step in the voltage clamp from ground to a positive value. At much lower concentrations both sucrose and glucose strengthened the binding of Ba 2+ to the protein, what in turn has the effect to shift the conductance voltage curve of the pore towards negative potentials. The possible mechanism underlying these effects and the analogies with other studies on the interaction of sugars and alcohols with proteins have been discussed.

The effects of osmolarity, calcium concentration and cell-size liposomes in the subphase on the surface tension of phospholipid monolayers were investigated. The monolayers were spread from chloroform solutions of phosphatidic acid at air/water solution interface. The liposomes (of average diameter 3 μm) were formed from phosphatidic acid/egg lecithin (1:2) mixtures in water or 0.1 ᴍ water solutions of sucrose. For this system there were critical concentrations of calcium ions to produce a large reduction of the monolayer surface tension. The threshold calcium concentrations depended upon the sucrose concentration in the subphase. Without sucrose the threshold calcium concentration was 8 mᴍ, while for isoosmotic sucrose solutions (0.1/0.1 ᴍ in/out of liposome) it was 14 mᴍ. It sharply increased to 28 mᴍ CaCl 2 at sucrose concentration difference across the liposome membrane 0.02 ᴍ and decreased to 26 mᴍ, 19 mᴍ, and 18 mᴍ with further increase of that difference to 0.04 ᴍ, 0.06 ᴍ, and 0.08 ᴍ, respectively. The rate of monolayer surface tension decrease was measured as a function of time at 30 mᴍ CaCl 2 and different sucrose concentrations in the subphase solution. The initial rates at first decreased with increasing the osmotic pressure and after that they increased. The minimum occurred at sucrose concentration gradient across the liposome membrane 0.02 ᴍ, i.e., at the point of maximum threshold calcium concentration required for large decrease of the monolayer surface tension. These facts may be explained by recent theories of dynamics of adhesion, instability and fusion of membranes modeled as thin films.

Incubation of synaptosomal plasma membranes (SPM) with liposomes of phosphatidylserine (PS), phosphatidylinositol (PIN) or phosphatidylglycerol (PGL), led to an increase of acetylcholinesterase (AchE) activity at concentrations of 0.1-1 μmol phospholip ids per mg SPM protein. The use of higher concentrations (1-7 μmol/mg protein), however, led to a progressive inhibition of the activity with respect to the maximal percentage of enzyme stimulation. To explain the enzyme stimulation by the acidic phospholipids, AchE was solubilized with the detergent Lubrol-PX and showed no change in the enzyme activity at any PS, PIN or PGL concentration used, indicating that these compounds do not act on the protein molecule directly. Arrhenius plots of AchE activities in untreated SPM (control), exhibited a break point at 23 °C , which was decreased to 16-17 °C in PS-treated SPM. Moreover, the Arrhenius activation energy (Ea) value in PS-treated SPM was increased related to the Ea below the break point in the control. These results indicate that acidic phospholipids do not act on AchE directly, but indirectly, affecting the membrane fluidity probably. Such modifications of interactions between lipid and AchE may control physiological processes in the central nervous system .

Cultures of vitamin D-deficient chick soleus muscle and 12 day-old chick embryo myoblasts were used to characterize the effects of 1,25-dihydroxy-vitamin D 3 and 25-hydroxy-vitamin D 3 on muscle cell Ca metabolism. Physiological amounts of both sterols increased the rate and extent of 45 Ca uptake by cultures. However. 1.25(OH) 2 D 3 was significantly more effective than 25 OHD 3 . The greater potency of 1,25(OH) 2 D 3 to increase Ca uptake could be shown after various treatment intervals of cultures and using a wide concentration range of both derivatives. Information about Ca pools affected by vitamin D 3 metabolites was obtained through kinetic analysis of Ca efflux in cultured myoblasts. Cytoplasmic and mitochondria Ca pools were identified on the basis of their half-times of desaturation and by selective inhibition of plasma membrane and mitochondrial Ca transport with LaCl 3 and Ruthenium Red, respectively. The data suggests that 1,25(OH) 2 D 3 acts on muscle cellular Ca by increasing Ca efflux and influx through mitochondrial and plasma membranes whereas the predominant effect of 25 OHD 3 is to increase Ca influx into mitochondria.

Native phytochrome (124 kdalton) was isolated from etiolated seedlings of Avena sativa L. cv. Pirol (Baywa, Munich). From low temperature spectra (0°C to - 165 °C) of phytochrome samples irradiated either with red or far-red light, spectra of intermediates were calculated. Both absorption bands of the phytochrome chromophore were reported. On the pathway P r → P fr , native phytochrome does not form a bleached intermediate (meta-Rb) in contrast to degraded phytochrome. An additional intermediate, meta-Rc (λ max = 725 nm), is formed instead. On the reverse pathway only two intermediates were found, lumi-F and meta-F. Oscillator strengths′ values were calculated and interpreted in terms of chromophore conformation. A scheme of intermediates for native phytochrome is presented.

The seven chlorophyll-protein complexes CPIa, CPI, LHCP 1 , LHCP 2 , CPa, LHCP 1 and LHCP 11 known in part also from the chloroplasts of higher plants were isolated from Chlorella fusca. They were characterized by their molecular weight, their absorption maxima and their ratio of chlorophyll a/chlorophyll b. The composition of the chloropyhll-protein complexes changes during the cell cycle of Chlorella fusca. The ratio of LHCP/CPI decreases at the beginning of the light period and the ratio LHCP/CPa after the 2nd hour of the light period. Both quotients increase at the 5th hour of the light period, have a maximum at the 8th hour of the light period and decrease afterwards during the second part of the cell cycle. These altera­tions are no reflections of chlorophyll-accumulation, but cause modifications in the organization of the thylakoids and influence the photosynthetic efficiency of Chlorella fusca. The size of the PSI- and PSII-units during the cell cycle was estimated by these changes of the LHCP/CPI- and LHCP/CPa-ratios. In addition evidence is given that the assembly of LHCP 1 and LHCP 2 is no simple association of the monomeric forms of LHCP I or LHCP II .

The cyanobacterium Anabaena flos-aquae (strain 1444) grown at different intensities of white light (900, 3500 and 30000 lux) showed changes in the content and composition of the pigments. Phycocyanin was more affected by high light conditions during growth than chlorophyll a. In comparison to in low white light grown cyanobacteria number of phycobilisomes and thylakoids decreased under strong light. A diminution of 14 CO 2 fixation, total amino acid content, glutamic acid and glutamine pools was found in strong white light grown cells. Under these conditions the majority of 14 C-labelling was measured in sugar phosphates. After pressure treatment a marked increase of 14 C-incorporation into amino acids could be obtained. Results were discussed with reference to regulation of buoyancy in Anabaena flos-aquae.

The fluorescence spectra of colonies of Propionibacterium acnes were studied under various experimental conditions. The spectra contained peaks at 580 nm and 620 nm. These bands were due to two different components; the 580 nm component was likely to be a metalloporphyrin, and there are indications that the 620 nm component could be a coproporphyrin. The 580 nm fluorescence was destroyed by the combined action of light and oxygen (no destruction under strict anaerobic conditions). A dark period interrupting the bleaching light stopped the destruction of this component for the time of the dark period. The initial production of the 620 nm component was due to the oxygen exposure. Upon light irradiation this component was later destroyed by the combined action of oxygen and light.

The effect of UV-B-radiation on PSII activity of spinach chloroplasts was analyzed by measuring the integrity of the herbicide-binding protein (HBP 32), by measurement of fluorescence induction in the presence of Diuron (DCMU), and by mathematical analysis of the fluorescence induction curves. It was shown that UV-B inactivates the PSII α-centers but not PSII β-centers. However, the possibility cannot be excluded that in addition the donor site of PSII near the reaction center is attacked by UV-B-radiation.

An enzymatic assay system for nitrate employing the membrane-bound nitrate reductase (EC 1.7.99.4) of E. coli is described. Contrary to previous enzymatic assay systems, the present method is a kinetic one, i.e. the substrate, nitrate, is assayed by measuring the reaction rate of the nitrate reductase-catalyzed reaction. Based on the observation that the nitrate reductase-catalyzed reaction obeys pseudo-first order kinetics, a test system is described allowing the assay of nitrate at a concentration as low as 1 ppm. The relatively high M ichaelis-M enten constant for nitrate (0.3 mᴍ) of the E. coli nitrate reductase favours nitrate assay by the kinetic method.

As reported in the literature, ferricyanide reduction of blue-green algal cells in the dark is linked to respiratory electron transport. This study has not verified this hypothesis. There was no concurrent activity of ferricyanide reduction by intact cells with respiratory oxygen uptake. It was found light-independent, showing little KCN-inhibition, but was strongly CCCP-sensitive. CCCP inhibition was reversed by KCN. - Both, respiration and ferricyanide reduction were stimulated by fructose which was actively accumulated by the cells (the apparent K m was 20 μM). Ferricyanide reduction was more sensitive towards dark starvation than respiration. Based on these data it is suggested that ferricyanide reduction and cyanide-sensitive respiration are parts of different electron-transport processes, competing for reduction equivalents originating from the same (endogenous) carbon pool.

From the ventral abdominal gland VII-VIII in male adult Leptoglossus australis, five volatiles have been identified by GC and MS data in comparison with authentic standard materials. Benzyl alcohol (31%) and 2-phenyl ethanol (42%) together accounted for over 70% of the total scent. The remaining materials identified were 2-octenyl propionate (19%), (E,E′)-2,4-hexadienyl acetate (6%) and (E)-2-hexenol (2%).

Mass spectrometric fragmentation patterns of alkyl-3,4-dihydro-2H-pyranes and alkyl-2,3-dihydro-4H-pyran-4-ones are described. Through GC/MS analyses, respective compounds showing unbranched carbon skeletons are identified for the first time as volatile signals of social and solitary bees and of the male moth Hepialus hecta L.

Rabbit antibodies against small peptides may be composed by subpopulations recognizing different epitopes made likely by few aminoacids. This explains the frequent crossreactivity of antipeptide antibodies with unrelated peptides. A suitable use of immunoadsorbents is suggested to obtain truly specific antibodies able to react with restricted amino acid sequences.