Band 39, Heft 9-10

A total of 138 samples of higher fungi, representing 35 species (15 belonging to the genus Amanita), were analysed for the trace element bromine using spectrophotometric and gas chromatographic methods. High concentrations of bromine, up to 100 mg/kg on dry weight, were encountered in Amanitaceae, especially in members of the subsections Phalloideae and Eu-Amanita, whereas in other fungi the mean value for this element was only 3,3 m g/kg. Some bromine-accumulating Amanitas also contained unusually high chlorine levels. Am axi­mum concentration of 3 percent on dry weight was measured in A. phalloides. It could be demonstrated that most, if not all of bromine and chlorine was present in the fungi as ionisable inorganic salts. Two organochlorine metabolites, previously reported in some mushrooms, were absent in A. phalloides and A. muscaria.

Cyanobacteria of the genus Microcystis have been found to liberate large amounts of β-cyclo- citral. While all seven strains of Microcystis studied possessed this property, it has not been found in any other cyanobacterial species. This chemical characteristic of the genus Microcystis enables the unequivocal systematic arrangement of controversial species. Another chemical property allows two chemotypes to be distinguished within the genus Microcystis: one exhibits an intensive release of isopropylmethyl disulfide, diisopropyl disulfide and diisopropyl trisulfide, substances which have not been previously found in any microorganism. The second chemotype excretes dimethyl disulfide and in some cases also dimethyl trisulfide and dimethyl tetrasulfide. Species producing isopropyl sulfides emanate an intensive sulphurous malodour.

A detailed investigation of non polar extracts of Meum athamanticum Jacq. rhizome (Um- belliferae) has led to the isolation of three phthalide derivatives. Sedanenolide, 6.6′,7.3′a-dili- gustilide and ligustilidiol have been identified by their spectral data. Furthermore, occurrence of such compounds seems to suggest assignment of intermediate biogenetic part to (Z)-ligustilide, the most accumulated phthalide in umbelliferous plants.

The 1000 x g organelles of Papaver somniferum latex have been shown to accumulate rapidly, large quantities of alkaloids (0.05- 1 mg/mg organelle protein), particularly morphine, thebaine, codeine and papaverine and have been found to exhibit a surprising degree of specificity for the accumulation of these alkaloids. The uptake of alkaloid is independent of temperature and does not show a requirement for ATP 10 -3 ᴍ, Mg 2+ 10 -3 ᴍ, or KCl 10 -3 ᴍ. It is unaffected by known ATPase inhibitors (chlormadinone acetate 2.5 × 10 -5 ᴍ, N,N-dicyclohexylcarbo- diimide 5 × 10 -4 ᴍ and sodium vanadate 10 -4 ᴍ) but showed considerable increase in morphine accumulation in the presence of 4-chloromercumbenzoate 5 x 10 -4 ᴍ, while N-methylmaleimide 5 × 10 -4 ᴍ had no effect, and carbonylcyanide 4-(trifluoromethoxy)phenylhydrazone 5 × 10 -4 ᴍ caused a reduction (ca. 30%) of morphine uptake. An acid medium, pH 4.5-5.5, also results in decreased morphine uptake.

Keywords The isoflavone conjugate biochanin A-7-O-glucoside-6″-O-malonate is degraded by Fusarium javanicum with an esterase to yield biochanin A-7-O-glucoside which is further cleaved by a glucosidase to the aglycone. Biochanin A is funnelled into a known catabolic sequence (Z. Naturforsch. 37c, 861 (1982)). Induction of the catabolism of p-methoxyphenylacetic acid is linked to biochanin A degradation, whereas p-hydroxyphenylacetic acid and 3,4-dihydroxy- phenylacetic acid degradation is substrate-induced.

Sixteen isoflavonoids have been isolated from the black gram. Phaseolus mungo L. (syn. Vigna mungo) following treatment with aqueous CuCl 2 . These include 12 known isoflavonoids: genistein. 2′-hydroxygenistein. 2′-hydroxydaidzein, kievitone, dalbergioidin, cyclokievitone. 5-deoxykievitone, 2′-hydroxydihydrodaidzein, isoferreirin, aureol, glycinol and demethylvestitol. In addition, kievitone hydrate, which was previously known only as a fungal metabolite of kievitone, was isolated together with 3 novel natural isoflavanones which were characterized as 4′-O-methylkievitone, cyclokievitone hydrate and 5-deoxykievitone hydrate.

Two c-type cytochromes (a high spin cytochrome c′ and a low spin cytochrome c-553(549) with asymmetrical α-band) and a low spin cytochrome b-558 from the purple sulfur bacterium Ectothiorhodospira shaposhnikovii were purified by ion exchange chromatography and gel filtration and characterized. Cytochrome c′ has a molecular weight of 33000 (determined by sodium dodecylsulfate electrophoresis), an isoelectric point at pH 4.5 and a redox potential of +37 mV. Absorption spectra show in the oxidized state maxima at 404 nm and in the range of 635 nm, in the reduced form maxima at 426.5 nm, 549 nm and a shoulder at 435 nm. The best purity index obtained was 0.48 (A 280 /A 426.5 ). Reduced cytochrome c′ reacts with carbon monoxide. Cytochrome c-553(549) has a molecular weight of 10400, an isoelectric point at pH 5.1 and a redox potential of +248 mV. The oxidized form shows the Soret-band at 410 nm. The reduced protein reveals an asymmetrical a-band at 553 nm with a shoulder at 549 nm, the a-band at 522 nm with a shoulder at 528 nm and the γ-band at 416 nm. The best purity index obtained was 0.18 (A 280 /A 416 ). Roth cytochromes could be isolated from the soluble fraction as well as from Triton X-100 treated membranes. Furthermore very low amounts of cytochromes c-553 and c-552.5 could be detected in detergent treated chromatophores. Cytochrome 6-558 - obtained from cells grown in the presence of reduced sulfur compounds in the medium - seems to be soluble or only weakly bound to the membrane. It has a molecular weight of 15800. an isoelectric point at pH 4.1 and a redox potential of -210 mV. The hemoprotein shows absorption maxima at 424.5 nm. 526.5 nm and 556.5 nm in the reduced form and at 416 nm in the oxidized state. The best purity index obtained was 0.26 (A 280 /A 42 , 4.5 ). In addition, there were hints for the occurrence of a high spin cytochrome b′. The cytochrome pattern as well as the amount of cytochromes were dependent on growth conditions.

Cell suspension cultures of Chenopodium rubrum accumulate high amounts of metabolically active glucose esters of p-coumaric and ferulic acid. Pulse-labelling experiments using [ 14 C]phenylalanine and application of the phenylalanine ammonia-lyase (PAL) inhibitor L-α-aminooxy-β-pnenylpropionic acid (AOPP) revealed that the glucose esters are subject to high turnover. A considerable portion of the ferulic acid was found as an insoluble component, probably bound to cell wall material. Application of m-fluoro-DL-tyrosine (MFT), an effective inhibitor of tyrosine biosynthesis via L-arogenate, markedly increased the amount of 1-p-coumaroyl-and 1-feruloyl-glucose accumulated.

The glutathione reductase from Escherichia coli strain S33 was purified to homogeneity by a simple and fast procedure consisting of two affinity chromatography steps. After 40-80% ammonium sulfate fractionation, the enzyme was adsorbed to an N 6 -2′.5′-ADP-Sepharose affinity column from which it was specifically eluted by a 0 - 10 mᴍ NADP + linear gradient. The enzyme was finally purified to homogeneity after a second affinity chromatography step in a C 8 -ATPR-Sepharose column, from which it was eluted by means of the same NADP + gradient. Starting from 182 g of E. coli cells. 6.9 mg of pure enzyme was obtained after a 2632-fold purifi­cation, with a total yield of 63%. The pure enzyme showed a specific activity of 361 U/mg, and its absorption spectrum was characteristic of a flavoprotein. with an A 272 A 450 of 7.84. The enzyme was a dimer with a molecular weight 109 000 and 40 Å hydrodynamic radius. The optimum pH were 7.5 and 4.5 with NADPH and NADH. respectively, as reductants. Apparent K′ m values of 16, 377, and 66 μᴍ were determined at pH 7.5 for NADPH, NADH, and GSSG, respectively. Upon storage the enzyme was stable at pH values ranging from 7.5 to 9.5, being additionally stabilized by FAD. NADP + , dithiothreitol, or glycerol. The pure enzyme was quite heat stable, denaturing signifi­cantly only after 10 min at 70 0 C. A marked activity loss was observed however, even at 0 °C, in the presence of 20 μᴍ NADPH. The enzyme was inactivated by low concentrations of para- hydroximercuribenzoate: the sensitivity towards such mercurial was greatly enhanced after reduction of the enzyme by NADPH.

Mercaptopyruvate sulfotransferase activity catalyzes the formation of pyruvate from mercaptopyruvate in the presence of suitable reagents as acceptor. It was detected in Lemna minor, Pisum sativum, Spinacia oleracea, Chlorella fusca, Synechococcus 6301, and Rhodopseudomonas palustris. Best activity was detected using dithioerythritol as a thiol reagent; good activity was obtained using mercaptoethanol, glutathione, mercaptopyruvate or sulfite as acceptor. The pH-optimum for the Chlorella mercaptopyruvate sulfotransferase was found around 9; the apparent K m for mercaptopyruvate was determined to 2mᴍ and for dithioerythritol for 5mᴍ using crude Chlorella extracts. The role of this enzyme is discussed in relation to cysteine catabolism by photosynthetic organisms.

The mutant strains A l a + and Y 5 of Rhodopseudomonas capsulata are defective in formation of the light-harvesting complexes B800-850 and B870, respectively, but synthesize the large bacteriochlorophyll-binding polypeptides of the respectively complexes, shown by amino acid composition, N- or C-terminal sequence and immunoblotting. The stable assembly of the light-harvesting complexes in the membrane seems to be dependent from the coordinated synthesis of all pigment and protein components.

A microsomal membrane fraction (6000 x g supernatant of a cell homogenate), isolated from coleoptiles of Zea mays, was separated by isopycnic sucrose density gradient centrifugation in the presence of EDTA and without a prior pelleting step to avoid irreversible sticking of different membrane species. The membrane fractions were characterized by assaying commonly used marker enzymes, and the levels of activity investigated of ATP hydrolysis, ATP-dependent H + transport, and co- and countertransport of ions, such as Cl - , fumarate 2- , K + and Li + . The following results were obtained: (1) ATP hydrolysis is performed by different enzymes associated with different membranes: - vacuolar acid phosphatase (AP; inhibited by molybdate); - Golgi phosphatase, revealing IDPase, pNPase and ATPase activity (not inhibited by molybdate); - ATPase activity of residual submitochondrial particles (sensitive to azide); - a H + -translocating ATPase at tonoplast membranes (Km (ATP) = 0.29 mᴍ; pH 7.5; stimulated by uncouplers and completely inhibited by NO - 3 ); - the H + -translocating ATPase of the plasmalemma (Km (ATP) = 0.39 mᴍ at pH 6.5, inhibited by vanadate, but not by NO - 3 ). The latter activity is evident only after an osmotic shock, indicating that PL vesicles primarily exist as inside-in-vesicles. (2) ATP-fueled H + pumps are localized at tonoplast (TO) and plasmalemma (PL) vesicles, they differ to some extent in their properties: (a) The PLH + pump has a very narrow pH optimum and exhibits highest levels of activity at pH 6.5 with a pronounced increase of activity between pH 7.5 and 6.5 (properties, obviously important in vivo for the regulation of active H + -extrusion by certain growth substances, which affect the cytoplasmic pH (Hager and Moser, Planta 1984, in press); in contrast, TO H + pumps show a considerably wider pH optimum with highest levels of activity around pH 7.5. (b) In variance to the PL H + pump the activity of TO pumps (Km (ATP) = 0.24 mᴍ) is regulated via the oxidation state of essential thiol groups. Their oxidation to the S -S - form (e.g. by blue light in the presence of a flavin) causes an inactivation, whereas a re-reduction by GSH or cystein restores the activity [51]. (c) The ATP-fueled H + transport into TO vesicles depends on an anion co-transport; most effective is Cl - , but there is also a stimulation by organic ions, C 4 and C 5 dicarboxylates, such as malate, succinate, fumarate, 2-oxoglutarate and aspartate; NO - 3 is inhibitory. (d) H + -transport into sealed PL vesicles is also anion dependent. In this case, however, NO - 3 is as effective as Cl - . (3) The TO membranes contain a H + /K + exchange mechanism responsible for a secondary active K + uptake into the vacuole. This mechanism could be the reason for a lower (ATP dependent) acidification of TO vesicles in the presence of K + compared with Li + . - Similar effects are observed with plasmamembrane vesicles, but in this case there is still the question whether a H + /K + -exchange, a K + channel, or both are acting.

Using a sensitive pH-difference spectroscopic method in combination with a three-column procedure of ion-exchange chromatography (overall yield 94%) the levels of cytochrome c in the large white butterfly, Pieris brassicae, were determined from the last larval instar to the adult insect. In the larva cytochrome concentration reached a maximum at mid instar and sub­sequently decreased to very low values in the pupa. During adult development the level of cytochrome c increased 50-fold in males and 43-fold in females; this sexual difference was expressed only after adult emergence. Biliverdin IXγ which occurs as a specific biliprotein complex was accumulated during the last larval instar and also in young butterflies. De novo synthesis of heme c, biliverdin IXγ and the corresponding apoproteins was demonstrated in newly emerged butterflies by injections of radiolabeled 5-aminolevulinate, lysine, leucine, and succinate, respectively. Cycloheximide inhibited labeling of both apoproteins and of heme c to 90% but that of the bilin to only 25%. This suggests that in cytochrome c but not in the biliprotein formation of the holoprotein depended on a coordinated synthesis of both constituents. In­corporation of 5-aminolevulinate into the biliprotein exceeded that into cytochrome c sevenfold indicating that biliverdin IXγ is the major product of the heme pathway in P. brassicae. The results are discussed in relation to the formation of mitochondria and flight muscles during postembryonic development of insects.

Incorporation of [ 14 C]5-aminolevulinate and [ 3 H]leucine into cytochrome c, biliprotein and total soluble protein was followed from the last larval instar to the adult stage in Pieris brassicae. The titer of ecdysteroids during the pupal stage was determined with a radioimmunoassay to correlate synthesis of heme products and of protein(s) with adult differentiation. Incorporation of both precursors showed a developmental profile with high synthetic activities in feeding larvae and in pupae after the release of ecdysteroids. Variation of the hormone titer during pupal life differed significantly in males and females. Labeling of cytochrome c by both 14 C and 3 H was as expected from the variation of its concentration reported in a preceding paper; highest in corporation was around adult emergence. The results demonstrate that i) the accumulation of cytochrome c in the developing adult insect is primarily due to de novo synthesis of both heme and apocytochrome c, performed under coordinate control, and ii) the concentration of 5-aminolevulinate is not rate-limiting in the formation of cytochrome c. Biliverdin IXγ, the major tetrapyrrolic product in this insect, seems to be directly derived from (free) heme and relatively short-lived as deduced from a time-course study. Formation of the bilin, i.e. destruction of heme, increased concomitantly to the initiation of adult differentiation by ecdysteroids in the pupa but later decreased at adult emergence. Synthesis of cytochrome c takes place as a late event during terminal development. Thus, the pathways leading to the two major heme products seem to be differently regulated during development.

Chromatin from two Syrian hamster tissues: the Kirkman-Robbins hepatoma and the liver, has been separated into soluble (S) and insoluble (P) fractions. Both fractions contain the complete set of five main histones but differ in respect of H1 subfractions. The hepatoma chromatin is known to contain an unusual H1 subfraction, H1 slow [12, 13], probably identical with a similar subfration present in hamster testes. The content of H1 slow in total H1 histone has been estimated for total, S and P chromatin from hamster hepatoma. The values 20.9 ± 7.2, 13.8 ± 1.8 and 26.8 ± 4.2%, respectively, were obtained.

The phospholipids and glycerides composition of spheroplasts of Mycobacterium smegmatis ATCC 14468 was examined. The percent total phospholipids in total lipids as well as cardio-lipin were found to be higher in spheroplasts as compared to their parent forms. Increase in cardiolipin and free fatty acids content and decrease in triglycerides levels were observed during spheroplasts formation. The results suggest that increase in cardiolipin content in sphero­ plasts is an adaptational change concomittant with the loss of cell walls.

Mixed Lipid/Protein Monolayers, Alveolar Surfactant, Monolayer Models Experimental data on surface pressure-surface area hysteresis of mixed serum albumin/dipal-mitoyl lecithin/sphingomyelin monolayers in the Langmuir trough are presented. Several possible physicochemical mechanisms of the hysteresis are discussed: Marangoni effect, surface pressure relaxations, bulk-to-surface diffusion interchange, and collapse. Depending on the concrete conditions each of these mechanisms can be important. Possible applications of these results to the alveolar dynamics are presented and discussed on the basis of the balloon model of the alveolus. The main conclusions of biological importance are that 1) the alveolar stability depends on the DPL/SM ratio as well as on the protein content. Under normal breathing conditions the surface pressure hysteresis is small and does not play a decisive role in the alveolar dynamics. 2) At large extent of compression the collapse predominates in determining the hysteretic behavior of the alveolar surface.

Electrofusion of mesophyll cell protoplasts of Avena sativa was induced by simulated atmospheric discharges. It is shown both experimentally and theoretically that the potential differences which occur at quite long distances from the point of lightning stroke are large enough to induce fusion. Besides electromagnetic waves which are emitted during lightning (G. Küppers and U. Zimmermann, FEBS Lett. 164, 323 (1983)) cell fusion may have also occurred directly by means of the voltage built-up on the earth during evolution in response to a lightning stroke.

The effect of extracellularly applied octopamine (10 -5 mol/l) on the receptor potential of the Limulus ventral photoreceptor was studied. Octopamine increases the sensitivity of the photoreceptor in the light-adapted state more than in the dark-adapted state. Extracellular application of phentolamine (10 -5 mol/l) has an opposite, antagonizing effect. The octopamine effect, though reproducible in the individual experiment, varies greatly from preparation to preparation.

The light induced transmission change (Arsenazo signal) of an Arsenazo III injected ventral photoreceptor cell of Limulus polyphemus was studied under voltage clamp. The transmission change which represents a change of free intracellular calcium ion concentration, [Ca 2+ ] i , was investigated for its dependence upon membrane voltage. The peak amplitude of the Arsenazo signal decreases in a linear fashion with the clamp voltage in the examined voltage range (from -80 to + 40 mV). In low Ca 2+ saline ([Ca 2+ ] e = 250 μᴍ) this decrease in the amplitude of the Arsenazo signal was more pronounced, while in saline with increased Ca 2+ ([Ca 2+ ] e = 40, 50 and 100 mᴍ), there is almost no change of the Arsenazo signal with varied membrane voltage. The recovery of the Arsenazo signal (i.e. recovery of the transmission back to the value before the light flash) is faster during hyperpolarization, this recovery being slowed down when the cell is depolarized. From these experiments it is concluded that a substantial part of the Arsenazo signal is due to a light induced influx of Ca 2+ from the extracellular space across the cell membrane into the cytoplasma. Conceivably the Ca 2+ could pass through light activated Na + channels. Subsequently the increased intracellular Ca 2+ is lowered to the preillumination level, by a membrane voltage dependent mechanism possibly an Na + -Ca 2+ exchange. The data do not exclude the possibility that a part of the Ca 2+ responsible for the Arsenazo signal is released from intracellular stores.

We established more than 200 primary cell lines of Cydia pomonella (codling moth). 81 of them were selected and screened for replication of two baculoviruses (from two different subgroups): the Choristoneura murinana NPV and the Cydia pomonella GV. Although all these cell lines had been derived from the same insect species, they varied largely in their response to challenge with the NPV. Most of them showed CPE or produced different amounts of poly-hedra. Interestingly, we also found a few cell lines that were permissive for GV replication. To our knowledge this is the first time that GV replication in cell lines has been obtained. Our results show that cell line properties are most important for baculovirus in vitro replication.

Abastract The production of C 6 -wound gases by cultivated and wild tomato plants (Lycopersicon esculenium Mill., L. hirsutum Humb. et Bonpl., L. peruvianum (L.) Mill., L. pimpinellifolium Mill., L. glandulosum C. H. Muller) and the tree tomato (Cyphomandra betacea (Cav.) Sendtn.) was investigated. The genuine mixture of compounds and the single compounds showed antifungal properties against the phytopathogenic fungi Botrytis cinerea Pers., Rhizoctonia solani Kühn, Fusarium oxysporum f. sp. lycopersici (Sacc.) Snyder & Hansen, Didymelia lycopersici Kleb, and Cladosporium fulvum Cooke.

The β 2 -dimer of tryptophan synthase from Escherichia coli exhibits weak binding of 8-anilinonaphthalene-1- sulfonic acid (ANS). Titrating the dye at 0.2 mᴍ concen­tration with the apo-β 2 -dimer at atmospheric pressure causes increased fluorescence emission at 480 nm (λ exc = 380 nm), corresponding to unspecific binding of the ligand to hydrophobic residues. Increasing hydrostatic pressure affects ANS binding. Up to 700 bar. a sigmoidal increase of ANS fluorescence reflects an increase in hydrophobic surface area, probably caused by subunit dissociation. At ~ 1 kbar, a maximum is reached; beyond this value, pressure competes with ligand binding causing fluorescence emission to be decreased again. Pressure release leads to a drastic fluorescence enhance­ment, ascribed to ANS binding to the partially and re­versibly denatured enzyme. Plotting the total fluorescence enhancement vs. pressure yields a profile which parallels the pressure dependent dimer ⇆ monomer transition monitored by subunit hybridization (T. Seifert, P. Barthol- mes, and R. Jaenicke, Biochemistry, in press).

A high performance liquid chrom atography method for determining the levels of 4-pregnene-3-ones in tissue containing fat, e.g. in thymus is reported. Following the extraction with chloroform/methanol the fat has been separated from the steroidal-fraction by using disposable extraction-columns. The steroidal-fraction has been analysed in two separation systems, on a RP 18 -column with a methanol/water gradient and on an amino column with a isopropanol/hexane gradient. The applicability of the described method is demon­ strated with calf thymus samples.

The effects of the administration of a single oral dose of vitamin D 3 to rachitic chicks on the in vivo incorporation of [ 3 H]leucine to proteins of skeletal muscle subcellular fractions was studied. A significant stimulation (50%) of labelling of mitochondrial proteins could be seen. The sterol affected the labelling of sarcoplasmic reticulum (20%) and contractile proteins (10%) to a lesser extent. These results confirm previous observations which im­plicate vitamin D 3 in the synthesis of mitochondrial proteins.

A new procedure for the isolation of highly purified acylamino acid amidohydrolase from hog kidney is d e­ scribed which allows the preparation of the enzyme with a recovery of about 45%, a 200 fold purification and a spec.activity of 350-500 U. The essential Zn 2+ of the enzyme was exchanged for Co 2+ , Ni 2+ , Mn 2+ and Cd 2+ , and the kinetic parameters K M , k cat and k cat / K M of the different enzyme species for a series of acetyl-L-amino acids were determined.

Mouse lymph node lymphocytes are irradiated at differ­ent dose rates and the B-cell receptors to anti IgG are tested. The expression of receptors is inhibited by irradia­tion. It is shown that the effectivity of irradiation increases with decreasing dose rate suggesting that membrane dam ­ age may be important for situations of chronic irradiation or implant radiotherapy.