Band 45, Heft 1-2

Several years of extensive research using the new powerful techniques of molecular biology have enabled the direct comparison of functionally or evolutionarily related genes and their products at the nucleotide and aminoacid sequence levels. Two types of synthase with similar functions are discussed as an interesting example. Stilbene synthases, e.g. resveratrol synthase, produce the stilbene backbone as a key reaction in the biosynthesis of stilbene-type phytoalexins. Chalcone synthase is a key enzyme in the biosynthesis of flavonoids, including certain phytoalexins derived from a 6′-deoxychalcone which is synthesized by cooperation of chalcone synthase with a reductase. Resveratrol and chalcone synthases utilize the same substrates (4-coumaroyl-CoA and 3 molecules of malonyl-CoA ) and catalyze the same condensing type of enzyme reaction (resulting in sequential addition of acetate units via malonyl-CoA ), but the products differ in the newly formed ring systems (resveratrol and naringenin chalcone). A comparative analysis of cloned DNA sequences and of the reaction mechanisms indicates that the two enzymes are closely related. It seems likely that the proteins possess a common scaffold for substrate recognition and for the condensing reaction, and that the different folding of an enzyme-bound intermediate prior to closure of the new aromatic ring is responsible for the formation of the different products. The same type of condensing reaction is utilized by the 2-ketoacyl-ACP synthases of fatty-acid biosynthesis. How ever, the available data indicate that these enzymes share little overall homology with either resveratrol or chalcone synthase. One exception maybe a short aminoacid sequence which corresponds to the active center of the condensing reaction in 2-ketoacyl-ACP synthases.

Ascorbic Acid Radicals as Indicator of Damage to Forest -EPR Investigations of Spruce Needles in vivo Hartmut B. Stegmann, Paul Schuler

Ricciocarpos natans grows best on Gamborg B5 medium supplemented with 2% sucrose. Six other mineral media were less effective on the growth. During a culture period, pH of the media changed depending on the ammonium and nitrate content of the respective media. In media of a molar ratio of ammonium to nitrate of 1:1 or 1:1.9, pH first dropped from 6.0 to about 3.5 and then rose again. In media where nitrate is the only nitrogen source or where there is a ratio of ammonium to nitrate of 1:12.5, pH rose during the first two weeks and after this dropped. Differences in the light intensity (2000 an d 6000 lx) and light regime (14 h light/ 10 h dark) had no effects on the growth. The cultures produced a variety of terpenoids. The same pattern of compounds was seen in field-collected material as well as in cultivated material. The content of the terpenoids decreased on nitrogen and phosphorous-deficient media.

The superoxide anion scavenging activity of 38 flavonoids, some of them isolated from Side-ritis mugronensis, Sideritis javalambrensis and Cayaponia tayuya were investigated by measurement of their inhibition of nitroblue tetrazolium reduction. Isoorientin, orientin, amentoflavone, leucocyanidol, eriodictyol, datiscetin and robinetin behaved as potent scavengers and structure-activity relationships were established.

Fifteen flavonoids have been isolated from the moss Bryum pseudotriquetrum . The new natural products kaempferol 3-O-galactoside-4 ′-O-glucoside, scutellarein 7-O-glucoside-6′′-malonyl ester, pigenin 7-O-neohesperidoside-6′′-malonyl ester and luteolin 7-O -neohesperi-doside-6″-malonyl ester were amongst them . Flavonols were found for the first time in mosses.

The volatile components of the essential oils of four Chinese species in the genus Asarum were investigated by means of GC-MS. Large amounts of elemicin, safrole, and 2-undecane to gether with several mono- and sesquiterpenes were identified in the essential oils. It was found that Chinese Asarum species is similar phytochemically to the genus Heterotropa in Japan.

Leaves from oak (Quercus robur) contain an acyltransferase that catalyzes the conversion of β-glucogallin (1-O-galloyl-β-D-glucose) to 1,6-di-O-galloylglucose, an intermediate in the biosynthesis of gallotannins. Substrate specificity studies revealed th at this enzyme was also active with several structurally related 1-O-phenylcarboxyl-β-D-glucoses; appreciable reaction rates, however, were observed only in the form ation of 1,6-di-O-protocatechuoyl-β-D-glucose. This to date unknown ester, as well as its digalloyl analog, was synthesized using acyltransferase immobilized on phenyl-Sepharose, and characterized by UV and 1 H NMR spectroscopy. In addition, the 1,2-di-O-β-D-glucose esters of benzoic and anisic acid were obtained in this investigation.

Maize (Zea mays L.) leaf phosphoenolpyruvate (PEP) carboxylase (PEPCase) (EC 4.1.1.31) showed a lag in activity when assayed after storage at 0-4 °C. The lag was promoted by high pH on storage (7.8 -8.5) and was observed over a range of assay pH (7.1 -8.5). Thermal reacti­vation of the cold-stored enzyme by assay temperature (18 °C) accounted for most of the hysteretic effect, but presence of PEP in the reaction mixture was required to completely eliminate the lag. Based on steady-state rates after the lag, stability of PEPCase in the cold was inde­pendent of protein concentration . It is suggested that low temperature and high pH induce a change in the oligomerization state of PEPCase, resulting in a less active but relatively stable form of the enzyme. The lag probably reflects a reversal of this process, promoted by assay temperature and presence of PEP.

Reactions of photolytically generated SO 4 ·̄ with 2′-deoxyuridine-5′-phosphate (5′-dUMP), uridine-5′-phosphate (5′-UMP) and thymidine-5′-phosphate (5′-dTMP) were studied by ESR spectroscopy in aqueous solution under anoxic conditions. From 5′-dUMP and 5′-UMP the 5′,5-cyclicphosphate-6-yl radicals 10 and 11 were generated (pH 2 -11) whereas from 5′-dTMP at pH 3 -8 the 5,6-dihydro-6-hydroxy-5-yl radical 14 and at pH 7 -1 1 the 5-methylene-2′-deoxyuridine-5′-phosphate radical 15 was produced. In the experiments with 5′-UMP in addition to radical 11 the signals of sugar radicals 12 and 13 were detected. It is assumed that the base radical cations act as intermediates in the SO 4 ·̄ -induced radical reac­tions. The 5′-phosphate group adds intramolecularly to the C(5)−C(6) bond of the uraclilyl radical cation whereas the thymidyl radical cation of 5′-dTMP reacts with H 2 O at pH < 8 to yield the 6-OH-5yl adduct 14 deprotonates at pH > 7 thus forming the allyl-type radical 15. In 5′-UMP transfer of the radical site from the base to the sugar moiety competes with intramolecular phosphate addition.

Purine nucleoside phosphorylase (PNP), from calf spleen, human erythrocytes and E. coli have been examined with regard to structural requirements of substrates and inhibitors. Kinetic parameters (K m , V max /K m ) for a variety of N(1) and/or N(7)-methylated analogues of guanosine, inosine and adenosine have been evaluated for all three enzym es. The substrate and/or inhibitor properties of purine riboside, 1,6-dihydropurine riboside, some deazapurine nucleosides: 3-deaza- and 7-deazainosine, 1,3-dideazapurine riboside (ribobenzimidazole), and a variety of acyclonu cleosides, have been determined with mammalian and bacterial enzymes. Overall results indicate distinct similarities of kinetic properties and structural requirements of the two mammalian enzymes, although there are some differences as well. The N(1) and O 6 of the purine ring are necessary for substrate-inhibitor activity and constitute a binding site for the mammalian (but not the bacterial) enzymes. Moreover, nucleosides lacking the N(3) undergo phosphorolysis and those lacking N(7) are inhibitors (but not substrates). Methylation of the ring N(7) leads to two overlapping effects: labilization of the glycosidic bond, and impediment to proton ation at this site by the enzyme, a postulated prerequisite for enzymatic phosphorolysis. It is proposed that a histidine interacts with N(1) as a don or and O 6 as an acceptor. Alternatively N(1)−H and C(2)−NH 2 , may serve as donors for hydrogen bonds with a glutam ate residue. The less specific E. coli enzyme phosphorolyses all purine ring modified nucleosides but 7-deazainosine which is only an inhibitor. On the other hand, the bacterial enzyme exhibits decreased activity towards N(7)-methylated nucleosides and lack of affinity for a majority of the tested acyclonu cleoside inhibitors of the mammalian enzymes. The foregoing results underline the fundamental differences between mammalian and bacterial enzymes, including variations in the binding sites for the purine ring.

The intermediate of chlorophyll biosynthesis, 5-aminolevulinic acid (ALA ), is a necessary prerequisite for the formation of protochlorophyllide (PChlide) and protochlorophyll (PChl) in the dark. The application of ALA to a dark-grown culture of the pigment mutant C-2 A′ of Scenedesmus obliquus increased the amount of PChlide 30-fold and the amount of PChl about 10-fold. The rates of ALA-dependent formation of PChlide and PChl reach their maximum values at different concentrations of added ALA . Similarly, the kinetics of PChlide and PChl formation in cells incubated with ALA are different. Cells of Scenedesmus mutant C-2 A′ incubated with various concentrations of ALA for different periods provide a good tool for future studies differentiating between PChlide and PChl metabolism . − The incorporation of Chl deriving from either PChl or PChlide into different pigment protein complexes is discussed.

Aspartate kinase (AK , EC 2.7.2.4) from the thermophilic, phototrophic prokaryote, Chloroflexus aurantiacus, was partially purified and separated from homoserine dehydrogenase (HSDH, EC 1.1.1.3). The molecular weights as determined by gel filtration were 130,000 and 46,000, respectively. HSDH had a moderately high thermal stability (50% inactivation at 84 °C) and displayed its activity optimum at 72 °C. By contrast, AK had its activity optimum at 52 °C (with a break-point in the Arrhenius plot at 42 °C) and was much less thermostable (50% inactivation at 67 °C). The K m -values for aspartate and ATP (determined in a pyruvate kinase-coupled test system) were 10.5 and 0.63 mM , respectively. The enzyme was strongly inhibited by L-threonine (K i = 10 μm) and activated by alanine, isoleucine, valine and methionine. L-Threonine acted as a mixed-type inhibitor in respect to aspartate, and non-competitively in respect to ATP. Contrary to AKs from Rhodospirillaceae, the enzyme from Chloroflexus aurantiacus was not subject to a concerted feedback inhibition by two amino acids of the aspartate family. The regulatory properties of the aspartate kinase are discussed in relation to the cellular amino acid concentrations.

The interaction between oppositely charged amino acids was studied by charge-transfer reversed- phase thin-layer chromatography. The dependence of the lipophilicity of Arg, Lys and Orn on the concentration of G lu , Asp, Gln and Asn in the eluent was considered to be related to the strength of interaction. The interaction of dibasic amino acids with Glu and Asp was stronger than with Gln and Asn. Mono- (Li + , Na + , K + , Rb + and Cs + ) and divalent (Mg 2+ and Ca 2+ ) cation s decreased the strength of interaction suggesting the electrostatic character of the interaction. Their inhibitory effect mainly depended on their concentration and to a lesser extent on the ion charge and hydrated ion radii. Stepwise regression analysis proved that the strength of interaction depends on the polarity parameters of amino acids and is independent of their chemical structure.

In the test system of isolated oat chloroplasts various structurally different cyclohexane-1.3- dione derivatives were investigated for their inhibitory effect on de novo fatty-acid biosynthesis. Cycloxydim proved to be the most efficient inhibitor in the group of the tested cyclohexane-1,3-diones. The alkoxyimino side-chain appears to be essential for the herbicidal activity. Compounds with variations in other substituents of the cyclohexanedione structure were less inhibitory. The I 50 -values of most of the applied substances for a 50% inhibition of de novo fatty-acid biosynthesis were in the range of 0.15 μM to 100 μM . Some compounds, however, showed no inhibitory effect.

A new class of non-adenylate anticytokinins, alkyl and phenyl N-phenylcarbamates and related phenyl N-pyridylcarbamates, has been developed. They have a structure that has an immediate resemblance to the non-adenylate class of cytokinins, N ,N′-diphenylureas, and N-pyridyl-N′-phenylureas, and the design of the molecule was made based on insight into the bioisoteric nature between the previous, non-adenylate s-triazine anticytokinins and the carbamate and urea structures. The activity in terms of the I 50 value of the most potent members, 4-fluoro- and 4-chlorophenyl N-(2-chloro-4-pyridyl)carbamates, was 0.3 - 0.5 x 10 -6 M when examined by the tobacco (Nicotiana tabacum L.) callus assay in the presence of 0.05 x 10 -6 M kinetin.

The influence of the oxime ether safeners, oxabetrinil and CGA-133205, on glutathione content, glutathione reductase (EC 1.6.4.2), and glutathione-S-transferase (EC 2.5.1.18) activity in seeds and seedlings of grain sorghum (Sorghum bicolor (L.) Moench. var. Funk G-522-DR) was investigated. Plant material for these experiments was derived from seed that was either untreated, or treated with 1.25 or 0.4g ai/kg seed of oxabetrinil or CGA-133205, respectively. Measurements were conducted at 0, 1, 2, 4, 8 , 12, 24, 36, and 72 h after the initiation of germination. In safener-treated sorghum seeds, the levels of total and reduced glutathione decreased gradually after the initiation of germination reaching a low at 8 to 12 h and then increased continuously reaching a maximum at 48 h. The greatest increases in glutathione levels were observed in oxabetrinil-treated sorghum seedlings. Glutathione reductase activity was consistently higher in untreated seeds of grain sorghum for the first 24 h of imbibition, but at 36 to 72 h glutathione reductase activity increased in the safener-treated tissues. CGA-133205 appeared to have a greater stimulatory influence on the activity of glutathione reductase than did oxabetrinil. For the first 8 h after germination was initiated, glutathione-S-transferase activity in oxabetrinil- and CGA-133205-treated seeds remained enhanced compared to that of untreated seeds, but was at or below the levels of the activity of glutathione-S-transferase extracted from untreated seeds for the remainder of the experiment. Non-enzymatic conjugation of metolachlor with reduced glutathione increased as the pH of the reaction solution increased from 6.0 to 8.0. Oxabetrinil at 1 to 40 μM and CGA-133205 at 1 to 160 μm enhanced nonenzymatic conjugation of m etolachlor with glutathione. At 80 and 160 |j.m , oxabetrinil reduced the am oun t of non-enzym atic conjugation of metolachlor. Oxabetrinil conjugated with reduced glutathione at low rates either enzymatically or non-enzymatically at pH 8.0 while CGA-133205 did not at any pH. In the presence of metolachlor, the amount of non-enzymatic conjugation of oxabetrinil was decreased, indicating that metolachlor is more reactive towards glutathione than is oxabetrinil. Overall, these data suggest that during the early stages of seed germination and seedling development of grain sorghum , oxim e ether safeners can enhance the detoxication of the herbicide, metolachlor, through enzymatic or non-enzymatic conjugation to reduced glutathione by enhancing either the level of reduced glutathione and/or the activity of glutathione-related enzymes.

Biochemie, Fachbereich Chemie, Universität Marburg, Hans-Meerwein-Straße, D-3550 Marburg, Bundesrepublik Deutschland Z. Naturforsch. 45c, Isocitrate Lyase c DNA, Malate Synthase c DNA, Glyoxysome, Leaf Peroxisome, Transition of Organelles Glyoxysomes are being replaced by leaf-type peroxisom es during the greening of dark-grown cucumber cotyledons. Light functions in this process as negative modulator of the gene expression of glyoxylate cycle enzymes but as positive regulator for the activation of glycollate oxidase formation. The differential gene expression was investigated at the level of m RNA amounts using c DNA probes hybridizing with malate synthase m RNA, isocitrate lyase m RNA, and glycollate oxidase m RNA. Hybrid ization probes were obtained from a c DNA library complementary to the germinationspecific m RNA s of cucumber cotyledons. The process of replacem ent of glyoxysomal proteins by leaf peroxisom al proteins was reversed to a certain extend when greened cotyledons were brought back in the dark. Hybridization on Northern b lots provided evidences that in greened cotyledons the amount of malate synthase m RNA and isocitratelyase m RNA starts to increase up on dark treatment.

The algal protists Reticulosphaera socialis Grell and Chlorarachnion reptans Geitler are “meroplasmodia”: They consist of separate cell bodies and a comm on reticulopodial network by means of which they capture and digest food organisms, for instance diatoms. When food becomes exhausted, the meroplasmodium disintegrates sooner or later into single cells. In Chlorarachnion reptans shortage of food may lead directly to globular resting cells with a distinct cell wall. In other cases disintegration is delayed. The plasmodial cells, still connected by reticulopodial strands, move about and join with single resting cells to form pairs. After pair formation they lose the reticulopodial connections, round off and form a cell wall of their own. The cells which rested from the beginning on start growing, whereas the cells which searched for them do not. It is probable that the nuclei of the latter are transferred into the growing cells so that synkarya are formed. If this the case, pair formation is a fertilization process and the growing cells are zygotes. A single zygote, isolated and transferred on a “lawn” of diatoms, gives rise to a plasmodium whose cells display both sexual types again.

Larvae of the arctiid moth Creatonotos transiens obtained each 5 mg of heliotrine, a pyrrolizidine alkaloid, via an artificial diet. 7 S-H eliotrine is converted into its enantiomer, 7 R 1 -heliotrine, and some minor metabolites, such as callimorphine. 7 S- and 7 R-heliotrine are present in the insect predominantly (more than 97%) as their N-oxides. The distribution of heliotrine in the organs and tissues of larvae, prepupae, pupae and imagines was analyzed by capillary gas-liquid chromatography. A large proportion of the alkaloid is stored in the integument of all developmental stages, where it probably serves as a chemical defence compound against predators. Female imagines had transferred substantial amounts of heliotrine to their ovaries and subsequently to their eggs; males partly directed it to their pheromone biosynthesis.

Enzymatically isolated myocytes from adult rats were used to measure 45 Ca-uptake from salt media of reduced Ca 2+ -content (0.1 mM) with normal (4 mM) or elevated (20 mM) potas­sium concentrations. Ca 2+ -uptake was interrupted by filtration followed by rapid chasing the filter with salt solutions containing no Ca 2+ , 2 mM Ca 2+ or 2 mM La 3+ . Rate and extent of 45 Ca-uptake of resting cells were found to be 3-fold enhanced when chas­ing was performed with La 3+ -containing media. In contrast La 3+ does not affect Ca 2+ -exchange of depolarized cells, the fluxes of which approximate rates sufficiently high for contraction activation and that are sensitive to Ca 2+ -channel blockers. The effect of La 3+ on resting cells suggests the existence of a small rapidly exchanging La 3+ -sensitive Ca 2+ -compartment lo­cated adjacent to the plasma membrane. This compartment is though to be either closed or has become La 3+ -insensitive in activated cells. The subsarcolemmal cysternae of the SR, the so-called “peripheral couplings” are most likely the morphological substrate of this compart­ment.

Hyperglycemia is an important independent risk factor in the development of coronary artery disease. Sixty one patients suffering from chronic and acute myocardial infarction out of which 12 patients were diabetic and 10 normal control subjects were investigated. The patients without diabetes and control subjects were subjected to oral and intravenous glucose tolerance tests. It was observed that the post load glucose level of most of the patients and fasting insulin level of patients without diabetes were higher as compared with control subjects. From the intravenous glucose tolerance test, half life of glucose and glucose assimilation coefficient were determined. Half life of glucose was increased significantly and glucose assimilation coefficient was decreased in the patients with myocardial infarction as compared with those of control subjects. The observations confirm that carbohydrate intolerance in myocardial infarction is not due to suppression of insulin secretion but due to peripheral utilization.

Incubation of deproteinized larval cuticle (chitin flakes) with purified arylphorin (calliphorin) or larval haemolymph of Calliphora vicina resulted in the formation of a chitin-protein complex. Enzymatic oxidation of N-β-alanyldopam ine (NDAB) in the presence of chitin flakes or the chitin-protein complex, resulted in various degrees of cross-linking of NBAD-quinone formed with chitin. This study confirms the involvement of arylphorin in the process of quinone tanning of insect cuticle.

Delayed light emission (DLE) from aggregated chlorophyll is used to probe energy transfer between aggre­ gated chlorophyll and β-carotene. Preilluminated β-carotene when injected into a dark chamber containing aggregated chlorophyll, induces DLE from aggregated chlorophyll. If the dark chamber contains only monomeric chlorophyll, there is no DLE. The intensity of DLE is dependent on the time of illumination of β-carotene. The decay of energy stored by the carotene is not a first order process. The first half life is about 2 min, the next about 10 min

A flavanone with 3′-OH as the sole B-ring substituent has been reported recently as a leaf and stem exudate constituent of Encelia stenophylla. The correctness of this unusual substitution is confirmed by synthesis of this flavanone.

Intracellular recordings of angular sensitivity from the photoreceptors of Aeschnid dragonflies (Hemianax papuensis and Aeschna brevistyla) are used to determine the magnitude and time course of acuity changes following alterations of the state of light or dark adaptation. Acuity is defined on the basis of the acceptance angle, Δρ (the half-width of the angular-sensitivity function). The maximally light-adapted value of Δρ is half the dark-adapted value, indicating greater acuity during light adaptation. Following a change from light to dark adaptation, Δρ increases slowly, requiring at least 3 min to reach its dark-adapted value. In contrast, the reverse change (dark to light) induces a rapid reduction of Δρ , and at maximal adapting luminances, this reduction takes place in less than 10 sec.