Band 36, Heft 3-4

Gaschromatographic, mass- and infrared-spectrometric evidence is presented for the identification of adenosine-3':5'-monophosphate (c-AMP) in maize seedlings (Zea mays). The genuine c-AMP content could be quantitated by GC-detection and refere to authentic c- AMP standards

This report describes the separation of components from a 4-coumarate: CoA ligase assay by means of liquid chromatography. With the aid of polyamide column chromatography it is possible to enrich and isolate chromatographically and UV spectroscopically pure p-coumaroyl- CoA using as a solvent 0.01% NH 4 OH in methanol subsequent to water and methanol alone. High performance liquid chromatography on octadecylsilane-bonded silica stationary phase allows a discontinuous determination of ligase activity. All components - ATP, Coenzyme A, pcoumaric acid, and the products AMP and p-coumaroyl-CoA - can be separated and accurately quantified within 20 min using a water-acetonitrile gradient, containing 1% phosphoric acid. The presented HPLC method may be used to affirm the accuracy of optical tests

Rutacridone-epoxide was isolated from roots and callus tissue cultures of Ruta graveolens L. and identified by 1 H-NMR and 13 C-NMR methods

Yellow coloration of pollen in some exceptional cases can be caused by the presence of intensely coloured flavonoids. Derivatives of herbacetin (sexangularetin) and of gossypetin (limocitrin) have been found as “yellow flavonols” in the pollen of some species of Paeonia and of Rumex. The pollen of Spinacea oleracea contains the 6-methyl ether of galetin and the quercetagetin derivatives patuletin and spinacetin

The pigment composition of Chlorella fusca has been investigated in the absence and in presence of vanadate and during iron stress. 1. Fe-deficiency generally decreases algal pigment content without change of the ratio chlorophyll a/b. 2. A twofold vanadate induced increase of the chlorophylls is accompanied by a similar enhancement of several xanthophylls, while lutein remains unaffected. 3. Vanadate stimulates the formation of β-carotene up to 5 fold, the effect being less obvious during iron stress

To study the flow of carbon through the shikimate pathway in tobacco cell cultures with low and high yields of cinnamoyl putrescines, the cell cultures were treated with glyphosate. In the presence of glyphosate the levels of free shikimic acid were increased more than 300-fold by both cell lines. Despite of a normally 10-fold higher level of cinnamoyl putrescines, the high yielding cell line accumulated only 25% more free shikimic acid than the low yielding cell line. This result together with earlier observations indicated that the increased formation of cinnamoyl putrescines was rather limited by the activity of phenylalanine ammonia lyase than by increased substrate supply caused by alterations in the shikimate pathway

Biosynthesis of phosphatidylcholine in young seedlings of Raphanus sativus is supplied with choline from degradation of the seed constituent sinapine (sinapoylcholine). This conclusion has been deduced from the following results: (1) Raphanus sativus seedlings accumulate approx. 70 nmol phosphatidylcholine which may be relevant for the consumption of choline, liberated during hydrolysis of approx. 130 nmol sinapine. (2) [ 14 C]choline and [ 14 C]ethanolamine, taken up by germinating Raphanus seeds, are exclusively consumed in the biosynthesis of phosphatidylcholine and phosphatidylethanolamine, respectively. (3) Pulse-chase experiments with seedlings at different germination stages show a reduced [ 14 C]choline incorporation into phosphatidylcholine at the time when degradation of sinapine occurs, obviously as a result of an isotope dilution by an increase of the endogenous choline pool. (4) After [ 14 C]choline pulse to immature seeds, during the process of seed maturation most of the activity taken up is incorporated into accumulating sinapine and approx. 50% compared to this into phosphatidylcholine. During seedling development the quantity of labelled sinapine rapidly decreases as a result of sinapine degradation with a concomitant label increase in free choline, phosphorylcholine, and phosphatidylcholine. Approx. 50% of the choline liberated from sinapine, is consumed in the biosynthesis of phosphatidylcholine. High performance liquid chromatographic analyses of phosphatidylcholine during Raphanus germination revealed that this phospholipid might be a metabolically active compound. Changes in the absorptivity of this compound at 210 nm indicate changes in the degree of unsaturation in the fatty acyl groups

When the abaxial epidermis was peeled from 5 to 6 day old oat primary leaves, and 3 cm segments were floated on radioactive phenylalanine or cinnamic acid solutions, more than 90 per cent of the radioactivity was incorporated within 3 to 7 h depending on the developmental stage of the leaf. C-glycosylflavones were labelled within 15 min and radioactivity in these compounds increased for several hours. Pulse labelling and pulse chase experiments with either phenylalanine or cinnamic acid, unequivocally demonstrate that oat flavones are stable end products of metabolism. However, this procedure does not distinguish between sequential biosynthesis of various flavones and their interconversion. Cinnamic acid was more efficiently (ca. 20 x) converted into oat leaf flavones than was phenylalanine, when the precursor was fed to leaf pieces, and flavones recovered from mesophyll protoplasts. Different labelling patterns were obtained with whole leaf segments and protoplasts which apparently reflect differences in tissue specific flavone biosynthesis of mesophyll and epidermis. Isolated mesophyll protoplasts incubated with [ 14 C]cinnamic acid synthesize 14 C-labelled flavones characteristic of the mesophyll, as well as several unidentified phenylpropanoid derivatives not found in the intact tissue. Data suggest that photosynthetically active mesophyll cells are a main site of tissue specific flavone biosynthesis

Using HPLC on a partisil column (5 μm) it was possible to separate two reaction products from an enzyme incubation of dimethylallylpyrophosphate and 3,6a,9-trihydtoxypterocarpan with a particulate fraction from elicitor-treated soybean cotyledons (Harosoy 63) or soybean cell cultures. These two products were identified by MS and 1 H-NMR analysis as 2- and 4-dimethylallyltrihydroxypterocarpan. Both compounds also occur as natural products in elicitor-treated soybean cotyledons and to a smaller extent in soybean cell cultures. Introduction of the dimethylallyl substituent into trihydroxypterocarpan increases the fungitoxicity against Cladosporium cucumerinum. Upon treatment with alkali, solutions of 3,6a,9-trihydroxypterocarpans turn to an intense red colour with λ max around 530 nm. One of the products from 2-DMA-THP could be isolated by reversed phase HPLC. According to its mass spectrum a quinoid structure is proposed for this red pigment

Several aminomethylphosphonic acids related to the herbicide glyphosate have been synthesised and their post-emergent herbicidal properties studied

The phototrophic bacterium Rhodopseudomonas palustris assimilated ammonium via glutamine synthetase and glutamate synthase. Diazotrophic and ammonium-grown cells had high levels of both enzymes, whereas enzymes of alternative assimilatory pathways were absent or had only low activities. Glutamine synthetase was purified to electrophoretic homogeneity within three steps by dye-ligand and ion exchange chromatography. Electron microscopy revealed a dodecameric molecular entity which was in accordance with parameters derived from electrophoretic techniques. The molecular weight of the enzyme monomer was 55 800; that of the dodecamer 670 000. The amino acid composition of R. palustris glutamine synthetase was determined and compared by a statistical method with other known enzyme compositions from prokaryotic and eukaryotic origins

1) Proliferation and energy metabolism of in vitro grown Ehrlich ascites tumor (EAT) cells in the presence of glucosone, (ᴅ-arabino-3.4.5.6-tetrahydroxy-2-oxo-hexanal) a competitive inhibitor of hexokinase, were studied. 2) Proliferation of the cells was completely inhibited by 2 mᴍ glucosone without severely affecting viability (dye exclusion test). No phase specific arrest of cell growth was observed. 3) Incorporation of [ 14 C]thymidine into an acid insoluble fraction of the cells decreases to 5% of the controls within 8 -10 h . Incorporation of [ 14 C]leucine begins to slow down immediately after treatment with glucosone. 4) The inhibitor (2 mᴍ) reduces the lactate production of the cells by 60%, respiration by about 20%; the ATP/ADP ratio slows down from 4.75 to 3.5. 5) The total inhibition of cell proliferation by 2 mᴍ glucosone cannot be explained exclusively by inhibition of hexokinase activity and impairment of energy metabolism. Because of a lack of specificity, glucosone is not a suitable inhibitor for studies on the relationship between hexokinase activity and cell proliferation of tumor cells

Sexual reproduction has a decisive function during the life cycle of all organisms. In the marine brown alga Cutleria multifida, multifidene (1), a low molecular weight olefinic hydrocarbon, has been recognized as a chemical messenger for gamete union. Comparative activity tests with 32 specially designed synthetic analogous of the natural attractant demonstrate the high specifity and low threshold concentration (6.5 × 10 -12 mol/L) of the gamete’s receptor system toward its natural signal (1). The findings establish the double bonds of multifidene (1) as the molecular coordination points in the messenger-receptor interaction. A tentative model is presented, which takes into account the evidence, that unpolar, but highly polarizable functional groups in the correct spatial arrangement are necessary for maximal mutual interaction

UHDBT (5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole) is an efficient inhibitor of photosynthetic electron transport in chloroplasts from spinach (pI 50 -value = 7.61) and the green alga Chlamydomonas reinhardii. At low concentrations of UHDBT its site of inhibition is located at the reducing side of plastoquinone, identical to that of 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU). This became evident from functional as well as binding studies. At higher concentrations of UHDBT a secondary inhibition site at the oxidizing side of plastoquinone, identical to that of 2,5-dibromo-3-methyl-6-isopropyl-1,4-benzoquinone (DBMIB) becomes evident

The partition of phylloquinone (vitamin K 1 ), of chlorophylls a and b and of the two main carotenoids, β-carotene and lutein, in subthylakoid particles (digitonin treatment) and chlorophyll protein complexes (sodium dodecylsulfate polyamide-gel electrophoresis) isolated from tobacco chloroplasts (Nicotiana tabacum L.) is described. 1. The “light particle” fractions (S 90 000, S 150 000) of digitonin fragmented chloroplasts are enriched in CP I and contain a higher proportion of phylloquinone, chorophyll a and β-carotene as compared to whole chloroplasts. This is visualized by high values for the ratio a/b (6 -8) and for β-carotene/lutein (1.7) as well as about 3 mol of K 1 per 100 mol of total chlorophyll. The “heavy digitonin particle” fraction (10 000 x g sediment), in turn, contains a higher proportion of chlorophyll b and lutein, but a lower level of phylloquinone than whole chloroplasts. 2. The chlorophyll a-protein CP I of pigmentsystem I, isolated by preparative gel electrophoresis using 0.5% and 4% SDS, is characterized by a stable level of phylloquinone (1 mol K 1 per 100 mol of total chlorophyll), high chlorophyll a/b ratios (7 -10) and high values for β-carotene/lutein (~ 6.0). The light-harvesting chlorophyll a/b-protein LHCP of photosystem II (chlorophyll a/b = 1.1 - 1.5, β-carotene/lutein = < 0.1) contains either low amounts of phylloquinone (0.5% SDS) or only trace amounts of K 1 (4% SDS). The free pigment fraction (FP) contains at 0.5% SDS 57% of the total phylloquinone of thylakoid membranes. At 4% SDS the K 1 amount in the free pigment fraction increases to 84%. 3. The phylloquinone partition studies in digitonin particles and SDS chlorophyll proteins indicate that there exist at least two localization sites for phylloquinone K 1 in the photosynthetic membrane. The CP I complex and a second site, presumably near photosystem II (CPa?)

From the water extracts of heat-treated spinach chloroplast lamellae, five compounds were isolated by column chromatography and identified by paper chromatography, UV-, IR-, 1 H-NMR- and 13 C-NMR spectrometry: besides the major component, sucrose, 1 glyophilized material contained 197 mg oxalic acid, 4.6 mg uridine and uracil and 22 μg lumiflavin. Only the isolated lumiflavin stimulated photosynthetic oxygen reduction and H 2 O 2 production by illuminated chloroplast lamellae. Evidence is presented that the stimulation of photosynthetic oxygen activation by water extracts of heat-treated chloroplast lamellae is most likely due to flavin derivative(s) (probably FAD) released from thylakoid-bound ferredoxin-NADP reductase by heat- and other treatments. Lumiflavin is artifactually derived from the solubilized flavin coenzymes during the isolation procedure

A thylakoid membrane preparation isolated from the blue-green alga Anacystis nidulans was freed from carboxysomes, soluble enzymes and the pigment P750 by floating in a discontinuous sucrose density gradient. In a buffer containing sucrose and the zwitterionic detergent Miranol S2M-SF the thylakoids were loaded on a linear 10-18% sucrose density gradient which also contained Miranol. The sedimentation yielded three bands, the lower two of which were green and the upper one was orange. The light green band in the middle of the gradient was the only one to show any photosystem II activity. This was measured as light-induced electron transport from diphenylcarbazide (DPC) to dichlorophenol-indophenol (DCPIP). The activity was sensitive to dichlorophenyl-dimethylurea (DCMU). The red absorption maximum of the particles in this middle band - henceforth called photosystem II particles - was found at 672 nm and the maximum of their low temperature fluorescence emission spectrum at 685 nm upon excitation with blue light. Cytochrome b 559 was the only cytochrome found in these particles; it was present at an average ratio of one molecule cytochrome per 40 -50 molecules chlorophyll a. C550 photoreduction with accompanying photooxidation of cytochrome b 559 was also observed in the photosystem II particles. Good photosystem II preparations did not contain any detectable amounts of P 700. By means of sodium dodecylsulfate polyacrylamide gel electrophoresis the polypeptide composition of the photosystem II particles was studied. Dissolution of the chlorophyll protein complexes was done under strongly denaturing conditions; consequently, no green bands were observed on the gels. The polypeptide pattern of the photosystem II particles showed two strong predominant bands of protein components with apparent molecular weights (app. mol. wts.) of about 50 000 and 48 000. These two bands are unique for photosystem II. Two other weaker bands were also found characteristic for photosystem II, the band of a polypeptide with an app. mol. wt. of 38 000 and that of a polypeptide with an app. mol. wt. of 31 000. Sometimes in addition the weak band of a polypeptide with the app. mol. wt. 27 000 was observed on the gel. The polypeptide 38 000 aggregated upon boiling of the sample in the presence of the denaturing agents prior to the electrophoresis, yielding an aggregate with an app. mol. wt. of 50 000. Additional polypeptides which were often found in the photosystem II particle preparation could be identified as subunits of the coupling factor of photophosphorylation CF 1 . None of the polypeptides described as characteristic for photosystem II are due to proteolytic activity. As the observed photosystem II activity was found to be DCMU-sensitive it appears that the DCMU-binding protein is among the here described photosystem II polypeptides. Moreover, the authors have reason to believe that one of the major protein components found characteristic for photosystem II is cytochrome b 559

Salmine A I, one of the components of the protamine from salmon, has been purified and characterized. The conformational preferences of salmine A I have been examined as a function of pH, added salts, presence of helix-supporting solvents, and temperature, using circular dichroism. It has been found that this small basic protein adopts predominantly an unordered conformation in aqueous solution. Addition of counter-ions, in particular perchlorate, and 2-chloroethanol induces to various extents the onset of the right-handed α-helical conformation. The results are discussed in comparison with those previously reported on the three main components of clupeine, the protamine from herring, and with the published conformational predictions by various statistical methods

Thin layers of synthetic homopolypeptides (poly-α-Ala, -Arg, -Asn, -Asp, -Glu, -His, -Lys and -Tyr) and proteins (myoglobin, concanavalin A, trypsin-inhibitor) were irradiated under solid state conditions in an electron microscope with 100 keV electrons. Radiolytic changes were investigated by amino acid analysis. The results are discussed in terms of the relative radiosensitivities of the constituent amino acids, and possible topochemical effects on the sensitivity pattern emerging. An attempt is also made to trace at least some of the predominant pathways of amino acid transformation, namely the production of alanine and a-aminobutyric acid

The ability of the high mobility group proteins (HMG-1,2,14 and 17) to serve as substrate for protein kinases was investigated by incubating them with a cytoplasmic and nuclear kinase. In both cases phosphate was incorporated into all four HMG proteins. The amount of phosphate incorporated and the specificity for the four proteins was quite different for the two kinases. Whereas the cytoplasmic kinase phosphorylated the HMG-1 and 2 to a higher degree than HMG-14 and 17, the nuclear kinase exhibited a high specificity for the HMG -17, leaving the other three proteins with only a small amount. The high preference of a nuclear kinase for HMG-17 may be indicative of a specific phosphorylation occuring also in vivo

The effect of interferon on the ESR spectra of erythrocytes treated with ascorbic acid has been investigated. This model system has been chosen since it represents identically the spectra obtained in cases with acute lymphatic leukemia. The data obtained show that small interferon concentrations increase, while larger concentrations decrease the effect produced by ascorbic acid resulting, finally, in the original erythrocyte ESR spectrum. Atomic absorption studies reveal the presence of copper which might be part of the active principle

Nuclei from hypodermis and midgut gland of the crayfish, Orconectes limosus, have been isolated with a slightly modified Chauveau-method. The isolated nuclei are well preserved and free from cytoplasmic contamination. They are able to incorporate [ 3 H]UTP into acid-insoluble material. This labelling is α-amanitin sensitive. The isolated nuclei are devoid of the mitochondrial/microsomal ecdysone-C-20-hydroxylase. The DNA: RNA: protein ratio is 1:0.8: 3.2 (using Triton X-100 during the isolation procedure the ratio is 1:0.4: 4.6) for nuclei from the hypodermis and 1:1: 3.8 for nuclei from the midgut gland. The absolute amount of DNA per cell is 1.6, resp. 3.5 pg. The final DNA-recovery is about 20%. The yield increases to about 60% if the final ultrazentrifugation step is omitted. The isolated nuclei bind 3 H-ecdysone and 3 H-20-OH-ecdysone specifically. As revealed by Scatchard analysis the affinity of the nuclear 20-OH-ecdysone receptor is 1-2 × 10 -9 ᴍ

A method for fitting of background-based curves is described whereas, with it’s aid, the peakpositions and areas in fitted spectrum sought, are easily located

From leaves of Holcus mollis L. the structure of the cyanogenic glycoside could be elucidated as 2-β-ᴅ-glucopyranosyloxy- 2S-butyronitrile (Heterodendrin) by means of chromatography, hydrolysis and 1 H-NMR-spectroscopy. By that another example for grasses is given where obviously the general assumed biogenetic pathway starting with tyrosine is not used

Among various Fusarium strains tested Gibberella fujikuroi (SAW) WR was shown to be a high producer of the phytotoxin fusaric acid

Acyclic terpenoid compounds, aldehydes, ketones, hydroxy esters, and dimethyl sulphone were identified in the dorsal exudate of the antelope Antidorcas marsupialis. The novel diterpenes α- and β-springene, found in this exudate, were synthesised from famesyl acetone

A series of straight chain ketones, saturated, unsaturated, and doubly unsaturated alcohols, saturated and unsaturated formiates, hexanal, formic acid, and a number of long chain fatty acids have been identified in the preorbital secretion of the grysbok, Raphicerus melanotis

The “Empirical Conformational Energy Program for Peptides (ECEPP)” by Scheraga and coworkers which calculates the conformational energies of given peptide conformations was adapted for use with the CGK TR440 computer system. New programs for energy minimization, statistical data analysis and graphical display of peptide conformations were added. In this way the range of application of the original ECEPP program was considerably extended. At the same time, its handling was greatly simplified by an improved data input/output

The effect of cortisol, dexamethasone, insulin and a liver cell growth promoting tripeptide on the secretion of plasma proteins into the medium of rat hepatocytes in monolayer cultures was studied. Cortisol and dexamethasone resulted in ~ 2.5-fold increase in the fibrinogen synthesis with general supression of album in and α-lipoprotein synthesis. On the other hand, insulin inhibited the biosynthesis of most plasma proteins except for the complement system and transferrin. Concentrations of α-lipoprotein, a-1-macroglobulin and haptoglobin were moderately elevated when the tripeptide Gly-His-Lys was applied in low concentration

A technique originally described for the isolation of Friend leukaemia virus envelope polypeptides [1] yields equivalent structures from Moloney leukaemia, AKR and BALB/c xenotropic virus as well as feline leukaemia virus. The envelope polypeptides are obtained as micellar protein complexes, named rosettes. Rosettes of the five m am malian type-C viruses examined are indistinguishable by electron microscopy. Separation of these aggregates in polyacrylamide gel electrophoresis under nonreducing conditions reveals a glycoprotein of about 85 000 d as their major component. Tryptic peptide analyses identify the viral origin of these polypeptides and emphasize strain specific differences in their primary structure