Band 37, Heft 7-8

ESR spectra of platinum uracil “blue” are discussed. Two main types o f paramagnetic species have been found, one of which can be explained by assuming an unpaired electron spin delocalized over d z² -orbitals of two equivalent Pt centers, the other one is attributed to a more localized d 7 Pt(III) center. In both cases the hyperfine structure suggests an additional smaller probability density of the unpaired electron at one adjacent platinum center. The smallest possible oligomers to realize this facts are tetramers in the first case and dimers in the latter one.

Roseanolone is a new diterpene from the African plant Hypoestes rosea (Acanthaceae). The isolation is described; the structure has been determined to be 1,2,3,3a,4,5,6,6a,7,8,9,10a-dodecahydro-5-hydroxy-3-isopropyl-3a, 6-oxa-6,9,10a-trimethyl-dicyclopenta[a,d] cycloocten-1-one by X-ray crystallography.

The isolation of 6-C-α-ʟ-rhamnopyranosylapigenin (isofurcatain) 7-O-β-ᴍ ᴅ-glucopyranoside, from Metzgeria furcata var. ulvula is described together with its identification. This is a new natural product, as also is isofurcatain.

The essential oil of strobiles from Humulus lupulus L. var. neomexicanus Nels. & Cockerell, grown in the semi-arid climate of Central Colorado, was isolated by steam distillation. A characteristic seasonal dependence of oil production was observed over two vegetation periods. - The main components of the essential oil were identified structurally by means of GC and GC-MS analysis. About 40 different compounds, including monoterpenoic hydrocarbons and alcohols, ketones, methylesters of saturated and unsaturated carboxylic acids were detected. Other sub­ stances were esters of short chain acids with monoterpene alcohols, plus sesquiterpene hydro­carbons.

Several introductionary reactions of isoflavone metabolism by Fusarium fungi are reported. Fusarium avenaceum hydroxylates formononetin in the 3′-position yielding 3′,7-dihydroxy-4′-methoxyisoflavone (calycosin). 3′-Hydroxylation of biochanin A to 3′,5,7-trihydroxy-4′-methoxyiso-flavone (pratensein) has been observed with Fusarium oxysporum f. sp. lycopersici and Fusarium oxysporum f. sp. lini. Depending on the growth medium Fusarium proliferatum metabolises for­mononetin either by 7-O-methylation or by 4′-O-demethylation.

The classic attempt to find optimum culture conditions lead to hormonautotrophic cultures and to dark grown cultures. We made the new attempt to rise the yield of rutacridone-epoxides by co­ culture with not hostspecific fungi. Conditions were chosen that only diffusible elicitors could in­ fluence callus tissue. 14 days of coculture mostly caused reduced acridone-epoxide content, growthrate and up to total disappearance of chlorophyll. Within 3 - 7 days of coculture some fungi induced a significant rise of acridone-epoxide content (up to 50-fold). Culture filtrates of these fungi showed comparable effects. Our results proved the acridone-epoxides to be phytoalexins. The use of fungal elicitors could be a new method to increase secondary product accumulation in tissue culture.

Two sulphur containing amino acids, dichrostachinic acid and djenkolic acid, and one sulphur containing imino acid, thiazolidine-4-carboxylic acid, have been isolated from the seeds o f Parkia speciosa Hassk. Dichrostachinic acid and djenkolic cyclic polysulphides in Parkia speciosa Hassk.

Three distinct glycosyltransferases have been isolated and partially purified from anthers of Tulipa cv. Apeldoorn. The following designations are proposed; UDP-glucose: flavonol 3-O-glucosyltransferase (GT-I), UDP-rhamnose: flavonol 3-O-glucoside rhamnosyltransferase (GT-II) and UDP-xylose: flavonol 3-glycoside xylosyltransferase (GT-III). The three enzymes exhibited an identical pH optimum within the range of 8.5 -9.0. The estimated molecular weight of GT-I and GT-II was about 40 000, GT-III showed a molecular weight of 30 000. GT-III required ions like NH 4 + or Ca 2+ whereas these ions have almost no influence on GT-I and GT-II activity. The enzymes have a slight requirement for SH-reagents, particularly DTE. As opposed to GT-II activity of GT-I and GT-III is significantly influenced by SH reagents and PCMB. Sucrose enhanced GT-III activity but only slightly GT-I activity, GT-II activity is not influenced. Flavonol aglycones can function as glycosyl acceptor for the GT-I, whereas flavonol 3-O-glycosides, luteolin, dihydroquercetin, naringenin, cyanidin, p-coumaric acid and some other phenols were inactive as acceptor. The best acceptors were isorhamnetin and quercetin (Km: 0.9 × 10 -6 ᴍ). GT-II did not accept aglycones as substrates. For this enzyme flavonol 3-O-glucosides were the most attractive substrates. GT-III did not have any affinity towards aglycones, too. This enzyme exhibited a high specificity for flavonol 3-O-glucosides as well as flavonol 3-O-galactosides. Both enzymes, the GT-II and GT-III, were able to glycosylate flavonol 3-O-diglycosides forming triglycosides. UDP-glucose (K m = 1.0 × 10 -4 ᴍ), UDP-rhamnose and UDP-xylose where shown to be the best glycosyl donors for GT-I, GT-II or GT-III respectively. The glycosyl transfer catalysed by the GT-I was shown to be a freely reversible reaction. In the whole anthers, highest specific activities of GT-I and GT-II were found during late stages of anther development. Similar results were obtained using the contents of anthers or the tapetum fraction. In contrary, high GT-III activity can be detected already in young stages of anther devel­opment. The highest activities of the three glycosyltransferases were found in the tapetum fraction, whereas the pollen fraction exhibited only poor activities.

Polynucleotide phosphorylase from cells of the cyanobacterium Synechococcus sp. has been purified 1400-fold by an improved procedure. The enzyme purified to homogeneity and lacking nuclease, phosphatase and protease contaminations reveals a single band of ADP polymerizing activity upon polyacrylamide gel electrophoresis under nondenaturing conditions which cor­responds to a molecular mass of about 275 000. The enzyme migrates as a single polypeptide of M r ≈ 70 000 when subjected to gel electrophoresis in the presence of dodecyl sulfate indicating a composition of α4 for the native enzyme molecule. The isoelectric point of the purified enzyme as determined by isoelectric focusing was found to be at 4.2 ± 0.1. Polynucleotide phosphorylase of Synechococcus is preferentially activated by Mg 2+ ; KCl has a significant stimulatory effect.

As a prerequisite for studies of the intracellular distribution of enzym es of the glycerol cycle in Dunaliella parva, the effect of the polycation DEAE-dextran on the permeability of membranes for various endogenous compounds and on photosynthesis was investigated. DEAE-dextran induces an increase of the permeability of membranes for low and high molecu­lar weight compounds: Under the influence of DEAE-dextran low molecular com pounds of the cells diffuse more rapidly along the chemical concentration gradient into the medium than macro-molecules. Furthermore enzymes of the cytoplasm do occur more rapidly in the outer medium than enzymes of the chloroplasts. Photosynthetic CO 2 fixation is inhibited already at low DEAE-dextran concentrations. This in­ direct inhibition is due to an unspecific efflux of compounds of low molecular weight, such as inorganic cations, metabolites or nucleotides. At high DEAE-dextran concentrations photo­ synthetic electron transport is directly affected at the level of the thylakoids: Electron transport is inhibited between plastoquinone and photosystem I. The efflux characteristics of marker enzymes of DEAE-dextran treated D. parva cells show that under optimal experimental conditions this technique may be a suitable tool to get information about the intracellular distribution of enzym es of unknown localization in the alga.

High- and low-affinity binding of photosystem II herbicides to isolated thylakoids of Spinacia oleracea and to intact cells of the unicellular green alga Ankistrodesmus braunii were investigated. Complete mutual displacement of bound diuron-type herbicides (e.g. diuron, atrazine, terbutryn) by either diuron- or phenol-type herbicides (e.g. ioxynil, dinoseb) in thylakoids as well as in intact algal cells was found for herbicide concentrations (< 4 nmol bound herbicide/mg Chl) which gave almost saturated high-affinity binding. This demonstrates a high degree of specific binding of these herbicides towards their receptor sites even in intact algal cells. In contrast, phenol-type herbicides are largely unspecifically bound in algal cells. The mechanism of binding of all photosystem II herbicides at the high-affinity (specific) binding site was found to be competitive. Within the group of diuron-type and of phenol-type herbicides as well as between these two groups, graphical and quantitative analysis of the Lineweaver- Burk plot and of the Dixon plot indicated competitive binding. From this a common binding site for both types of herbicides was concluded. The involvement of two different herbicide binding- proteins is discussed. Low-affinity (unspecific) binding was found to be irreversible in contrast to the easily reversible high-affinity binding. Irreversibility was indicated by a lack of displacement. It is proposed that low-affinity binding represents either a partitioning of the herbicides into the lipophilic parts of the membranes or an attachment to distinct receptor sites. Unspecifically bound herbicides might be responsible for several high concentration effects of the photosystem II herbicides, which are described in the literature. Evidences for the possible existence of a second binding site of these herbicides are presented.

DCMU, bentazon, amitrole and SAN 6706 affected the formation of the pigment-protein complexes and caused drastic alterations in the absorption of light and in the transfer of the absorbed energy in the antennae systems. Bentazon and DCMU, photosystem II inhibitors, did not change the pigment absorption and fluorescence excitation spectra. After application of both herbicides the long wavelength fluorescence emission band at 740 nm was reduced similar as in young developing leaves. Although DCMU and bentazon inhibit the photosynthetic electron transport at the same site, bentazon mainly suppressed the formation of the photosystem I complexes CP 1 a and CP 1 while DCMU mainly reduced the photosystem II complex CPa. Bentazon specifically enhanced the formation of LHCP3. This may be important for the increased grana stacking in plastids from bentazon treated plants. The bleaching herbicides amitrole and SAN 6706 inhibited the formation of carotenoids leading to an accumulation of lycopene, phytofluene and phytoene, while the accumulation of chlorophylls was suppressed. This bleaching effect was most pronounced during growth under higher intensities of light. In weak light (100 lux) amitrole reduced the long wavelength fluorescence maximum but the fluorescence excitation was not affected. With amitrole at 2000 lux and SAN 6706 at 100 lux the long wavelength emission band was further decreased and the fluorescence excitation spectra point to a less efficient energy transfer to chlorophyll a. The fluorescence spectra changed due to herbicide treatment resembled those of not yet fully developed leaves. In contrast to the photosystem II herbicides the bleaching herbicides amitrole and SAN 6706 had a similar effect on the formation of pigment-protein complexes. After growth at 2000 lux both herbicides suppressed the formation of the photosystem I complex CP 1 a and the photosystem II complex CPa. At 100 lux only the formation of CPla was affected. Except for DCMU all herbicides assayed primarily changed the formation of photosystem I.

The effect of DCMU, bentazon, amitrole and SAN 6706 on the formation of chloroplast pigments and quinones was investigated using plants that were grown in total darkness or continuous white, red or far-red light. All herbicides assayed affected the formation of chlorophylls, carotenoids and quinones but DCMU had only minor effects. Like for chlorophylls and carotenoids the formation of quinones was most suppressed in plants grown in the presence of the herbicide in continuous white or red light, but the effect on the formation of quinones was much lower as compared to the pigments. The observation that the biosynthesis of quinones is still maintained in SAN 6706 treated bleached plastids which are lacking chlorophylls and carotenoids indicates that quinones are synthesized at the plastitd envelope and stored in the osmiophilic plastoglobuli. Amitrole and SAN 6706 induced a strong chlorosis. It was of particular interest that chlorosis was also induced by the photosystem II inhibitor bentazon. DCMU was not effective. The inhibitor concentration for 50% inhibition in the chlorophyll and carotenoid content was 5 × 10 -6 ᴍ for SAN 6706,3 × 10 -4 ᴍ for amitrole and 10 -3 ᴍ for bentazon. As already reported by others SAN 6706 treated plants accumulated phytoene in large amounts. The highest phytoene content was observed in plants that were grown in the dark. Amitrole treated plants accumulated lycopene. But in addition other carotenoid precursors like phytoene and phytofluene were also accumulated. In contrast to phytoene lycopene was only accumulated in plants that were grown in the light. Particularly for SAN 6706 and amitrole the expression of the bleaching effect was depending on the light intensity and light quality that was used during plant growth. The herbicide effect ewas predominantly expressed at higher light intensities and after irradiation with red light. The observation that the induction of chlorosis is very sensitive to red light as compared to white or blue light is suggesting that phytochrome is involved in the development of the herbicide toxicity. It also supports that in SAN treated plants chlorophylls are photodecomposed directly by light because of the lack of photoprotecting carotenoids but mainly β-carotene in these plastids. Further support for this was given by the demonstration that SAN treated plants which were grown at very low light intensities turned green and were photosynthetically active.

The effects of a series of 2-arylamino-1,4,5,6-tetrahydropyrimidines on photosynthesis in spinach chloroplast were studied. The compounds proved to be uncouplers of photophosphorylation, while inhibition of electron transport occurred at higher concentrations. These effects were rather irreversible and the activity increased with lipophilicity. Inhibition of electron transport could not be reversed by trypsin treatmeni. Loss of membrane integrity might be the underlying mechanism of these effects.

A particle-bound, Mg 2+ -dependent ATPase activity is investigated in cell-free extracts of the tyanobacterium Anabaena variabilis. The enzyme can be clearly distinguished from the cyanobacterial coupling factor of photophosphorylation and from the alkaline phosphatase. It requires low concentrations of Ca 2+ for maximal activity and is inhibited by ortho-vanadate, indicating that the enzyme may form a phosphorylated intermediate in its catalytic cycle. The distribution of the ATPase in sucrose density gradients does not follow that of thylakoids. It is concluded from these characteristics that the enzyme is bound to the plasma membrane. The cytochrome oxidase activity of the extracts appears to be restricted to the thylakoids.

The sarcoplasmic reticulum in situ is an intricate tubular network that surrounds the contractile material in striated muscle cells. Its topographical relationship to other intracellular components, especially the myofibrils, is rather rigidly mainiained by a cytoskeleton which enmeshes Z line material and sarcoplasmic reticulum and, ultimately, is anchored at the plasmalemma. As a result, the two main components of the sarcoplasmic reticulum, the junctional SR and the free SR, retain their typical location in the A band region and in the I band region, respectively. The junc­tional SR, which is thought to be the site for calcium storage and release for contraction, is, thus, always well within one micron of the regulatory proteins associated with the actin filaments. The junctional SR, a synonym for terminal cisterna applying to both skeletal and cardiac muscle, is generally held to be involved in the translation of the action potential into calcium release, mainly because of the close topographic apposition between the junctional SR and the plasmalemma, especially in skeletal muscle. This attractive structure-function correlation is challenged by the observation that in bird cardiac muscle 80% of the junctional SR is spacially far removed from plas­malemma, the site of electrical activity. This anomalous topography is not in conflict with the notion that translation of the action potential into calcium release may be accomplished by a dif­fusible transmitter substance, e.g. calcium. Any hypothesis dealing with this problem must ac­ count for the anatomy of the bird heart.

The changes in intracellular sodium activity and contractility produced by short-lasting application of a fast-acting cardiac glycoside were measured in guinea-pig ventricular muscle. It was found that under certain conditions the change in twitch tension paralleled the change in sodium activity. It is suggested that the electrogenic sodium pump may be involved in the regulation of both the mechanical and the electrical activity of cardiac muscle.

At 0.1 mg/ml protein and 0.45 µᴍ free Ca 2+ 1 mol trichloroacetic acid precipitable phosphate is incorporated into 100,000 g SR protein as hydroxilamine sensitive acylphosphate. At nearly physiological protein concentration (ca. 7 mg/ml) a total of ca. 0.8 mol phosphate/100,000 g protein is incorporated, from which a fraction of 0.3 mol/100,000 g protein is insensitive to the hydroxylamine treatment, i.e. it is alkylphosphate. Phosphorylase kinase accelerates the alkylphosphate incorporation ca. 3-fold and enhances its final level to 0.7 mol/100,000 g protein. At 1.6 nM free Ca2+ alkylphosphate incorporation occurs at high SR concentration to a maximal extent of 0.5 mol/ 100,000 g protein. The incorporated alkylphosphate is present in comparable amounts in the 100,000 Mr Ca 2+ -transport ATPase and a polypeptide of M R 9,000.

A number of equilibrium and kinetic measurements are presented to characterize the partial reactions of the ATPase and transport cycle in sarcoplasmic reticulum vesicles. The cycle begins with calcium and nucleotide binding on sites available on the outer surface of the vesicles. A phosphorylated enzyme intermediate is then formed, and the calcium sites are subjected to a change in their orientation and their affinity for calcium. It is shown that steps involved in calcium release on the inner side of the vesicles are rate limiting for the cycle, and are followed by hydrolytic cleavage of the intermediate with release of inorganic phosphate and recycling of the enzyme.

The effects of ATP-analogues on Ca 2+ -ATPase and (Na + + K + )-ATPase have been studied. The participation of sulfhydryl groups in the recognition of ATP by both transport ATPases is indicat ed by the fact, that the disulfide of thioinosine triphosphate inactivates both enzymes. The reactivity of rapidly and slowly reacting sulfhydryl groups in the ATP binding sites of both enzymes is altered by the presence of transport substrates. At least in (Na + + K + )-ATPase Na + and Mg 2+ appear to alter the structure of the ATP binding site, which conclusion is fortified by the fact, that the photoinactivation of the enzyme by 3′-O-[3-(2-nitro-4-azidophenyl)-propionyl]-ATP need Mg 2+ . Chromium(III)ATP, a MgATP analogue, inactivated both transport ATPases by the formation of a stable chromo-phosphointermediate. In the case of Ca 2+ -ATPase this was concomited by the occlusion of Ca 2+ in a stable form. No occlusion of Na + was observable so far in the (Na + + K + )-ATPase. Contrary to the expectation of the Albers-Post-scheme the hydrolysis of the phosphointermediate formed from chromium(III)ATP was protected by K + , but activated by high concentrations of Na + . Consequently, despite of the inhibition of (Na + + K + )-ATPase activity chromium(III)-ATP supported the Na + - Na + -exchange reaction in everted red bood cells.

In single skeletal muscle Fibres perchlorate causes a large shift of the potential dependence of contraction activation to more negative potentials without a corresponding alteration in the kinetics of the inactivation process.

The Ca uptake of the SR of a skinned fibre, which was studied with an indirect method, shows a biphasic time course. A total Ca concentration between 8 and 23 mᴍ was accumulated in the SR by an initial fast process (τ ≈ 4.7 s). This Ca-uptake activity of the SR is high enough to bring about relaxation o f twitch contraction in vivo. The magnitude of a subsequent slow process (τ ≈ 10 3 s) indicates that the total Ca storage capacity of the SR of skeletal muscle is in the order of 60 - 75 mᴍ.

Using x-ray microanalysis and cryoultramicrotomy, calcium and other diffusible elements were localized in heart muscle strips which had been shock frozen under different, defined conditions of electromechanical coupling. Guinea pig papillary muscles were shock frozen: 1) 1 1/2 seconds after paired stimulation, 2) 5 minutes after rest in normal bath medium and 3) 5 minutes after rest in bath medium to which noradrenaline was added. In 1) high calcium concentrations of 11.5 mmol/kg d.w. were regularly detected in sites at the level of Z-lines, which probably corre- spond to the Z rete of SR. In 2) in which the mechanogram of the first contraction after rest normally showed a small and retarded peak, the cell stores seemed to be nearly empty, with exception of a few regions between the mitochondria which revealed calcium accumulations of 77 mmol/kg d.w. These regions included JSR and/or T-tubuli. In 3) in which the mechanogram of the first contraction after rest normally showed a retarded peak with high tension, calcium was found in several cell structures. The highest amount, 25 mmol/kg d.w., was detected over the cell membrane. Measurable amounts were also detected over Z-lines and sarcomeres. In the present experiments, the respective rate of rise of tension, and time to peak tension, were extremely different. Possible correlations between different contraction patterns and different calcium stores involved in the various experiments have been discussed.

The farinose frond exudate of the fern Notholaena aschenborniana was previously shown to contain a mono­ acetate and a mono-butyrate of a 8-hydroxyflavonol as major constituents and the same pair of esters of a second 8-hydroxyflavonol as minor components. Now we have been able to identify the 8-acetates of two further flavonols. The structures of 5-hydroxy-3,7,2′,3′,4′-pentamethoxy-8-acetoxyflavone and 5,4′-dihydroxy-3,7,3′-trimethoxy-8-acetoxyflavone have been elucidated by spectroscopic methods. Both are novel natural products.

After treatment with the fungus Helminthosporium carbonum or aqueous copper sulphate, excised Lathyrus sativus leaflets produce pisatin and a second isoflavonoid phytoalexin (lathycarpin) identified as (+)-6aR; 11aR-2,3-dimethoxy-6a-hydroxy-8,9-methylenedioxypterocarpan.

Marked anomalies, previously unresolved, exist in the CD spectra of oligoadenylates and oligouridylates at the pentamer or tetramer stage, indicating that these molecules differ in conformation from the preceding and the following member of the series. The CD of oligoadenylates retains a positive Cotton effect even at 80 °C. Caution must be exerted when predicting oligonucleotide structures from CD spectra.

A glycoprotein of an apparent molecular mass of 46000, gp 46, was enriched by affinity chromatography from the virus-and cell-free culture medium of adult T-cell leukemia virus (ATLV) infected cells, gp 46 was specifically precipi­tated with sera from patients with adult T-cell leukemia known to react with the adult T-cell leukemia associated antigen (ATLA). Thus, gp 46 is a novel component of the ATLA antigen complex.