Band 37, Heft 1-2

A new antibiotic, 1 -hydroxy-2-nonyn-4-one (1), has been isolated from submerged cultures of several strains of Ischnoderma benzoinum. The compound exhibits high inhibitory activity against yeasts and filamentous fungi at concentrations of 1-5 μg/ml. DNA, RNA, and protein syntheses in cells of the ascitic form of Ehrlich carcinoma are strongly inhibited by the same concentrations.

A series of polyunsaturated vinyl-hydrocarbons (n-C 15 to n-C 17 ) (10, 11, 12, 15, 16 and 17) including the as yet unknown all-cis-1,7,10,13-hexadecatetraene (15) as well as some rare sesquiterpenes such as isocomene (8) or modhephene (7) have been isolated from the root oil of Senecio isatideus. Unambigous positioning of the double bonds of the polyolefines was easily established by 13 C-NMR spectroscopy [16]. A new mass spectroscopical method utilizing NO as a reactant gas for chemical ionization [1] made possible the reliable identification of minor and trace constituents without any chemical pretreatment or further derivatisation.

The uneven distribution of carotenoids and chlorophylls between several chlorophyll- carotenoid-proteins isolated from radish chloroplasts by SDS-polyacrylamide-gel electrophoresis is described. Lutein and neoxanthin are enriched in the light-harvesting chlorophyll a/b-protein LHCP 3 , which exhibits low chlorophyll a/b ratios (1.1-1.3) and high values for the ratio chloro­phyll a/β-carotene (a/c = 60-180). β-carotene is bound not only to the chlorophyll a-protein CPI + CPIa of photosystem I, but also to the chlorophyll a-protein CPa. Both chlorophyll a-proteins are characterized by high values for the ratio a/b and low values for the ratio a/c.

[ 14 C]β-Carotene was fed to juvenile stick insects, Carausius morosus, of the fifth instar. Radioactivity was incorporated into 2-hydroxy-, 2-oxo-, and 3,4-didehydro-2-oxo-carotenoids of the β,β-type. These transformations are due to the insect’s own capacity; any contribution by microbial symbionts can be ruled out. A study on the labelling kinetics clearly shows that the biogenesis of hydroxy- and oxo-carotenoids is correlated to a decrease in the carotene precursor, but only up to mid instar. Thereafter, oxidation of the carotene is very low but the transformations of its metabolites continue as before. Predominantly β,β-carotene-2,2'-diol is dehydrogenated to 3,4,3',4'-tetradehydro-β,β-carotene-2,2'-dione via two hydroxyketones. This discontinuous utilization of β-carotene could be due to a stop at mid instar either in the oxidation or in the absorption in the gut of this precursor.

Enzyme preparations from flowers of Verbena hybrida do not only catalyse hydroxylation of the B-ring of flavanones and dihydroflavonols in the 3'-position but also in the 5'-position. Enzyme activity for 3',5'-hydroxylation was found to be localized in the microsomal fraction and required NADPH as cofactor. Evidence is provided that the formation of the 3',4',5'- hydroxylated flavanone (5,7,3',4',5'-pentahydroxyflavanone) and dihydroflavonol (dihydromy- ricetin), respectively, proceeds via the corresponding 3',4'-hydroxylated compounds eriodictyol and dihydroquercetin, respectively, which are most probably formed by action of the same enzyme. Enzyme activity for 3',5'-hydroxylation was found to be strictly correlated with the prescence of 3',4',5'-hydroxylated flavonoid compounds in the flowers.

Ankistrodesmus braunii NAD(P)H-nitrate reductase is a complex hemoflavomolybdoprotein composed by eight similar subunits. The flavin prosthetic group, identified as FAD, is essential for the NAD(P)H-dependent activities of the complex, and is located before the heme chromo- phore in the enzyme electron transport chain from reduced pyridine nucleotides to nitrate. Fluorescence studies indicate that nitrate reductase can dissociate about 80% of its FAD by incubation at room temperature, the flavin dissociation being followed by a parallel decrease of NADH-nitrate reductase activity. Dissociation of FAD from the protein is easily increased by dilution or prolonged dialysis of the enzyme preparations. However, exogenous FAD specifically prevents the dissociation of enzyme-bound flavin, and protects the NAD(P)H-dependent activities. The K m for FAD, as a protector of NADH-cytochrome c reductase activity, is 4 nᴍ. In addition, dithioerythritol also prevents the flavin dissociation, and therefore the presence of free sulphydryl groups in the FAD-domain is suggested. FAD-depleted nitrate reductase, obtained by several methods, is unable to recover its original activity when incubated in the presence of FAD alone or with thiols.

Excitation of isolated chloroplasts in the presence of ferredoxin and NADP by repeated short flashes yields a polyphasic absorption change at 700 nm. Assuming first-order reactions, the signal may be resolved into three distinct components with average relaxation times of approxi­mately 20 μs, 150 μs and 20 ms. Their relative magnitude is dependent on experimental conditions; their spectral characteristics indicate that all three components may be ascribed to P-700. Concurrent measurements of Y-NADPH, the flash yield of NADP reduction with an enzymatic recycling method, allowed Y-NADPH to be compared to the magnitude of each of the three P-700 components and to total P-700. In general, the data show a good correlation of NADP reduction with the sum of the μs-phases but not with the ms-phase or total P-700. Analysis of light intensity curves (blue or far red flashes) with a mathematical model which yields maximum values for all parameters at infinite light intensity shows that in both cases approximately two moles of the microsecond component of P-700 turn over for each mole of NADPH formed. In contrast, the molar ratio of the ms-component to the yield of NADP reduction is approx. 0.2 in blue and approx. 6.3 in far red light. The data suggest that only that portion of the P-700 pool which relaxes in the microsecond range may be involved in the reduction of NADP while the ms-component is funtionally isolated from linear electron transport.

It has been proposed that the mutagenic activity associated with the ascorbic acid autoxidation system may involve hydrogen peroxide and peroxide radicals. We report here that the mutagenic effect may also partially reside in as yet unknown secondary products. The observed mutagenicity of 2,3-diketogulonic acid, one of the main oxidation products, reflects its ability to form hydrogen peroxide.

The fungal metabolite mevinolin, known to be a potent hypocholesterolemic agent, excerts in vitro a strong inhibitory effect on microsomal HMG-CoA reductase from etiolated radish seedlings at a concentration of about three magnitudes lower than the K m towards the natural substrate (S)-HMG-CoA (I 50 = 2.5 × 10 -9 ᴍ). Beside this, mevinolin significantly inhibits the root elongation of radish as well as of wheat seedlings already at low concentrations of 10 to 100 ppb (= 2.5 × 10 -8 to 2.5 × 10 -7 ᴍ).

From the pressure dependence of 1 H high resolution NMR spectra of two dipeptides (glycylsarcosine and N-acetyl-ʟ-proline-NH-methylamide in the range 0.1 MPa ≤p ≤ 150 MPa the activation volumes ⊿V ≠ for the amide rotation are derived. This conformational transition is characterized for glycylsarcosine by ⊿V ≠ = 4 ± 1 cm 3 · mol -1 and for. the proline derivative by ⊿V ≠ = 7.5±1 cm 3 · mol -1 . From the given results the maximum contribution of proline cis ⇌ trans isomerisation to the pressure dependence of the rate of reactivation of proteins can be estimated to ~ - 30% per MPa and proline present.

This paper reports Rayleigh scattering experiments on metmyoglobin crystals and freeze dried myoglobin which was exposed to air with different partial pressure of water vapor. While dry myoglobin shows no fluctuations between conformational substates such “breathing modes” are rearly seen in water covered myoglobin. Larger amounts of water increase the average mean square displacements. In crystals the dynamic behaviour is hindered by the crystal packing. The results are analysed by a theory describing the motion within the molecule by a Langevin equation with restoring forces corresponding to a square well potential.

The aggregation of bovine ribonuclease (RNase) induced by either ·OH or Br •̅ 2 radicals has been studied. These radicals, which were generated by pulse radiolysis of aqueous solutions with 16 MeV electrons, readily attack the enzyme thus producing RNase-radicals which subsequently combine. At initial radical concentrations low enough (≤ 0.1 radical per enzyme molecule) to prevent multimerizations other than dimerization, rate constants for the latter process were determined from the increase of the light scattering intensity after the pulse: 2 k 2 = (2.2 ± 0.3) 10 6 l/mol s (Br •̅ 2 initiated dimerization) and (5.4 ± 0.4) 10 6 l/mol s (·OH initiated dimerization). The G-values for dimerization are: 1.3 (Br •̅ 2 ) and 0.85 (·OH). Transient optical absorption measurements revealed the existence of phenoxyl radicals of tyrosine (TyrO·) that decayed with a rate constant of 2 k 2 (total) = (5.5 ± 0.7) 10 6 l/mol s (Br •̅ 2 )-case). The difference between k 2 and k 2 (total) presumably indicates the occurrence of disproportionation.

8-Methoxypsoralen (8-MOP) in ethanol, acetone, benzene, or CCl 4 is photoxidized under UV- irradiation (320-400 nm). Photoxidized 8-MOP (O 2 -8-MOP) is stable in the organic solvents, but it is destructed in water or in liposome suspension. The destruction rate constants are 0.04 s -1 in water and 0.004 s -1 in liposome membranes as estimated by the kinetics of the chemilumin­escence accompanying the destruction. In course of O 2 -8-MOP destruction the residues of phospholipid unsaturated fatty acids are oxidized. Generation of the singlet oxygen ( 1 ⊿ g ) by excited 8-MOP is observed neither in acetone, nor in ethanol. Quantum yield of 1 ⊿ g formation in CCl 4 is less than 3%. A pattern is proposed for 8-MOP-sensitized oxidation of unsaturated lipids proceeding without direct attack of lipids by singlet oxygen.

The thylakoid differentiation in the endosymbiotic cyanelle Cyanocyta korschikoffiana depends on the developmental stage of the whole symbiotic association Cyanophora-Cyanocyta. During culturing under permanent light the intrinsic or extrinsic location and the amount of the chlorophyll-protein complexes and of the phycobilines in the thylakoids change markedly. During this modification of the thylakoids the reaction centers of phytosystem I and II are more and more protected by other proteins. Moreover the amount of phycobilisomes increases in relation to the amount of the chlorophyll-protein complexes. The energy transfer becomes more and more efficient. All these aspects show the typical behaviour of a mass culture of a free-living blue-green alga and imply that Cyanocyta korschikoffiana is a symbiotic cyanelle much more than a chloroplast-like organelle.

A fluorescence spectroscopy method is described for studying association of RNA polymerase with DNA templates. Using double beam differential fluorescence at excitation and emission wavelengths of 285 and 335 nm, respectively, the new technique discriminates non-specific decrease of fluorescence intensity by addition of DNA from quenching of polymerase fluorescence by protein-nucleic acid interactions. Comparing the results with studies of UMP incorporation into RNA, the K s -values of template-binding were in good agreement with the values for RNA synthesis, pointing to specific interaction of polymerase and the DNA template measured by the fluorescence method. While E. coli enzyme showed higher affinity for templates such as heat- denatured poly [d(A-T)] and poly [d(G-C)] parsley RNA polymerase I accepted such templates with the same affinity as salmon sperm DNA. It is obvious that divalent cations are not necessary for the interaction of both enzymes with single-stranded DNA-templates. UMP incorporation studies suggest that transcription is a cooperative process.

Properties of Ca 2+ - or Ba 2+ -dependent transfection and transformation in Escherichia coli were examined, using ΦX174 replicative-form (RF) DNA and plasmid DNA. For the transfection and transformation, a heat pulse step was dispensable and the yield of transfectants was, in most E. coli strains, rather reduced by the heat treatment. The heat pulse step was also detrimental for the transformation of certain strains such as lipopolysaccharide mutants. The first stage of the DNA uptake process (formation of DNA · recipient cell complex) was dependent on low temperature and Ca 2+ ion. A substantial amount of the complexed RF-DNA was released from the bacteria, by washing with a chilled Tris buffer. Although a RF-DNA · cell complex was formed even at 37 °C or in chilled 0.05 ᴍ MgCl 2 , the complex did not yield transfectants.

Photorespiratory activity was measured in entire plants of five tobacco variants. These tobacco variants are: the green type N. tabacum var. John William’s Broadleaf (su/su Aur/aur or su/su Aur/Aur) the chlorophyll-deficient tobacco mutant Su/su (Su/su Aur/Aur) and the chlorophyll- deficient mutant Su/su var. Aurea (Su/su Aur/aur). Furthermore, two recently characterized phenotypes originating from N. tabacum var. Consolation namely “consolation green” (Aa Bb) and “consolation yellow-green” (aa bb). In entire plants of these phenotypes photorespiration was measured as 18 O 2 -uptake in the light. This uptake was compared with the enhancement of CO 2 - fixation in the Warburg effect i. e. when the oxygen partial pressure is lowered from 21% O z to 3% O 2 . The principal conclusion from these measurements is firstly that under the assay conditions which are identical for all 5 phenotypes (330 ppm CO 2 , 14000 lux white light and 25 °C) all five phenotypes yield considerable differences in photorespiratory activity. Furthermore, we were able to show that in the different phenotypes the global O 2 -uptake in the light is repartitioned to different degrees among different metabolic pathways. Thus, in JWB which is under the assay conditions the only fast growing species, only half of the measured 18 O 2 -uptake belongs to glycolate metabolism or photorespiration proper, the other half belongs to a Mehler type reaction in which excess reducing power is eliminated apparently already at the level of photosynthetic electron transport. In the chlorophyll-deficient mutant Su/su, however, the observed 18 O 2 -uptake in the light belongs under the assay conditions exclusively to glycolate metabolism (no Mehler type reaction). The chlorophyll-deficient mutant Su/su var. Aurea behaves more like JWB, that is, part of its 18 O 2 -uptake is due to a Mehler type reaction and only the remainder is involved in CO 2 - metabolism, which has been already found out previously by genetic analysis. In addition photorespiration depends in Su/su more on the temperature than in the other phenotypes tested. One of the implications of our results could be that it makes a difference to the plant whether excess reducing power is disposed of at the level of the photosynthetic electron transport chain (via a Mehler type reaction) or at the level of CO 2 -metabolism.

Radiation-induced polymerization method was applied to the immobilization of yeast cells. The effects of irradiation, cooling and monomer, which are neccessary for polymerization, were recovered completely by subsequent aerobical incubation of yest cells. The ethanol productive in immobilized yeast cells increased with the increase of aerobical incubation period. The growth of yeast cells in immobilized yeast cells was indicated. The maximum ethanol productivity in immobilized yeast cell system was around three times as much as that in free yeast cell system.

Nanochlorum eucaryotum was isolated from a sea water aquarium housing different sponge species, cucumarias, small crustaceans and annelids. This bright green marine alga differs from all other known coccoid species. Its most prominent features are its very small cell size (1.5 pm) and its reduced cellular organization. Its cell contains one nucleus, one chloroplast, one mitochondrium and small vacuoles. Sometimes a Golgi apparatus can be seen. No other subcellular features have been observed. The cell wall is thin and smooth and does not contain any material of high electron density; only dividing cells show a rougher surface. The cells split into two daughter cells. No sexual reproduction has been observed in this organism. We have analyzed the ultrastructural cell organization, the amount of total DNA and RNA per cell, the pigment composition, the growth requirements and the sensitivity towards different inhibitors (chloramphenicol, cycloheximid, penicillin, lysozym and cellulase). The results afford the introduction of a new species; a new family or suborder of coccoid green algae is discussed.

Nanochlorum eucaryotum is a very small species of unicellular coccoid green algae (1.5 pm). The growth of Nanochlorum under different conditions of salinity, pH and light intensities was studied. Optimal growth rates were observed with normal sea water salinity and low light conditions at pH 7.0. The contents of chlorophylls, carotinoids, soluble proteins and the chlorophyll a to chlorophyll b ratio were measured. The light saturating curves of Nanochlorum cells grown under light intensities of 100 lx, 2000 lx and 10000 lx reveal a very narrow capacity of light adaptation. When cultured under higher light intensities, Nanochlorum was not able to reach high photosynthetic activities but underwent a photoinhibition of photosynthesis. The contents of cytochrome f, P-700 and ribulosebisphosphate carboxylase were low and comparable with those of low light adapted Chlorella cells. The analysis of the chlorophyll-protein complexes shows that about 80% of total chlorophyll is bound in the light harvesting chlorophyll protein complexes. All results indicate that Nanochlorum is a low light adapted marine organism with very narrow ecological flexibility.

When unilamellar vesicles are prepared in the presence of 3-Palmitoyl-7-oxy-coumarin (abbreviation 3P-UBF) this lipoid pH-Indicator is anchored by its fatty acid chain to the membrane and can be used to measure pH-changes at the outer and inner membrane surface (ranging from pH 5 to pH 9.5). By rapidly-changing the pH of the outer aqueous phase a pH- gradient is set up across the vesicle membrane. The rate of the subsequent H + or OH - influx into the vesicles can be measured as a change of the 3P-UBF absorbance at 424 nm. This was done with a stopped-flow-spectrophotometer at temperatures between 10 °C and 50 °C. Suspensions of vesicles prepared from egg-lecithin or ʟ-dipalmitoyl-lecithin were investigated in buffered salt solutions. The influence of Na + , K + , Cl - , SO 4 2- and valinomycin on the rate of absorbance changes was studied at different temperatures. It was found that the rate of the pH-equilibration between the aqueous phase outside and inside the vesicles depends on the direction of the pH-gradient. This new result together with a high H + /OH - permeability of vesicle membranes found in recent studies from other laboratories and confirmed by this investigation is interpreted to indicate a higher permeability of the vesicle membrane to OH - -ions compared to H + -ions. (Calculated values are: P OH = 1.4 × 10 -4 cm/s at pH 9 and P H = 8.3 × 10 -7 cm/s at pH 5 and 20 °C.) All data described in the literature in detail agree with this suggestion but a pH-dependence of P OH and P H cannot be excluded.

By comparing proton and electron spin resonance spec­tra of twofold and single oxidized ascorbate and acetylated derivatives therefrom it can be concluded that semidehy- droascorbate as well as dehydroascorbate are of bicyclic structure due to the formation of a furanose ring of their side chain.

Hydroxyrutacridone epoxide was isolated from roots and callus tissue cultures of Ruta graveolens L. and identified by spectroscopic methods by comparison to rutacridone epoxide.

Flavonol 3-O-methyltransferase activity has been de­tected in Calamondin orange peel and root tissue, tobacco cell culture and young shoots of Chrysosplenium americanum. The enzyme was purified from the latter tissue by precipitation with ammonium sulphate, ion-exchange chro­matography and finally by chromatofocusing on Poly- buffer exchanger at pi 4.8. The focused enzyme exhibited strict stereospecificity towards quercetin, MW of 65000 daltons and pH optimum of 7.5-8.5. The apparent K m values for quercetin and S-adenosyl-ʟ-methionine were 1.4 and 70 μM, respectively.

In buffer solution (pH 7,4) ruthenocene and osmocene are far more stable than ferrocene. The metallocenes ruthenocene and osmocene are metabolized by microsomes of mouse liver in the presence of NADPH and O 2 . The K M -values are similar for both metallocenes, the rate of metabolism is higher for osmocene that for ruthenocene.

Neutral Red, Chromatin Fluorescence, Nucleic Acid Cytochemistry, Intercalative Binding to DNA After treatment with neutral red at low concentrations (10-6, 10-5 m), the chromatin o f chicken erythrocytes shows an intensive red fluorescence, which is reduced or prac­ tically abolished when nuclei become stained by using the dye at concentrations higher than 10~* M. Both the fluores­ cence and staining reactions are dependent on the DNA content of chromatin. Neutral red fluorescence o f nuclei increases considerably after treatment with inorganic cations (Al3+, Ba2+), while previous treatments with meth­ ylene blue reduce the fluorescence intensity. The possi­ bility that chromatin fluorescence depends on the inter­ calative binding of neutral red is suggested.

Three different polyvalent proteinase-inhibitors have been detected in the albumin gland of the snail, Achatina fulica and identified by means of fibrin-agar immunelectrophoresis. The inhibitory activity of these substances against several proteinases from different sources was investigated.