Band 48, Heft 7-8

Bacterial enzyme systems, especially those which are involved in cell energetics, often show a common characteristic feature: their constituents (either interacting enzymes or subunits of a given enzyme complex) are physically separated. They are located in different functional enti­ties, such as cytoplasm or periplasmic space. This kind of cellular and macromolecular organi­zation enables the cell to establish spatially separated but neighbouring zones in which distinct conditions are created or maintained. This intrinsic imbalance is one of the keys for the process of life. As the mediator between the two compartments, the cytoplasm and the periplasmic space, the cytoplasmic membrane -itself a functional entity -not only acts as a barrier, but carries a set of functional enzyme components, thus contributing to the interaction between compartments. Examples to illustrate this concept are enzyme systems involved in anaerobic glycine metabolism, aerobic utilization of carbon monoxide, proton or sodium translocation across the membrane, and intracellular hydrogen cycling used by the cell for the generation of a proton gradient.

Both phenylbutazon and mofebutazon inhibit oxidative fragmentation of the methionine derivative, 2-keto-4-methylthio-butyric acid (KMB) by xanthine oxidase - or diaphorase me­ diated OH radical production. Differentiation of the two non-steroidal antiinflammatory drugs is possible by means of determining oxygen reduction by xanthine oxidase or diaphorase in the presence of the naphthoquinone, juglone, where only mofebutazon shows an inhibitory effect.

A new hirsutane derivative, phellodonic acid (1), has been isolated from fermentations of Phellodon melaleucus strain 87113. Its structure was elucidated by spectroscopic methods. The compound exhibits antibiotic activities towards bacteria and fungi. 1 is the first bioactive metabolite from cultures of a species belonging to the family Thelephoraceae.

An isoflavanoid derivative has been isolated as a phytoalexin from intact plants and cell suspension cultures of Ornithopus sativus after treatment with CuSO 4 , yeast elicitor, or a spore suspension of Colletotrichum trifolii. Based on its physical and spectroscopic properties the substance was identified as glabridin. After induction, the transient in crease in the content of this phytoalex in was preceded by a transient increase in the activities of PAL and CHS.

FPLC of crude cell extracts of Chlorella kessleri reveals two protein fractions with phosphoenolpyruvate carboxylase activity. Their mole masses, 955 kDa and 543 kDa , suggest that they are dimer and monomer of the same enzyme. Further data, how ever, indicate that they are more likely two different iso-forms of phosphoenolpyruvate carboxylase: Attempts to interconvert both proteins in vitro, such as through treatment with NaCl, metabolites and thiolor histidine-group effecting reagents, were unsuccessful. The substrate affinity of the large protein was slightly higher than that of the small protein (K m = 1.13 and 0.76 mᴍ, respectively); and the sensitivity to enhanced temperature was more pronounced in the large than in the smaller protein (half lifes = 23 min and 55 - 60 m in, respectively). Some properties of both fractions, however, proved identical: 1. pH optima at pH 8.5 - 9,2. Hill coefficients approx. 1,3. no significant regulatory effect of glutamine, glutamate, aspartate and malate, 4. increase in Km and in Hill coefficient by citrate, and 5. identical behaviour in ion exchange chromatography. Functions and localization of the assumed two iso-forms of phosphoenolpyruvate carboxylase remain to be clarified.

Two different O-glucosyl transferases (F3GT and F7GT ) catalyzing the transfer of ᴅ-glucose from UDP-ᴅ-glucose to the 3 and 7 positions of flavonol, respectively, were isolated from the young leaves of Paederia scandens var. mairei. F3GT and F7GT, which were recovered in about a 1:1 ratio in the activity, were purified by about 140- and 136-fold, respectively, by precipitation with ammonium sulfate followed by ion exchange chromatography and chromatofocusing. F3GT and F7GT both had a pH optimum of 7.5 in Tris-HCl buffer, and an M r of 43 kDa. Neither F3GT nor F7GT had a Mg 2+ requirement. Both were inhibited by each 1 mᴍ of Zn 2+ , Cu 2+ , N-ethylmaleimide and p-chloromercuribenzoate, and both were stimulated by 14 mᴍ 2-M E . F3GT and F7GT were different from each other in having an isoelectric point (pI) at pH 5.12 and 4.50, respectively. F3GT mediated the transfer of D-glucose exclusively to the 3-hydroxyl group of kaempferol and some flavonols, but neither the 7-O-glucosides nor the 3-O-glucosides of their flavonols were able to accept D-glucose. On the other hand, F7GT mediated the transfer of D-glucose exclusively to the 7-hydroxyl group of kaempferol and some flavonols, and in addition, the 3-O -glucosides of kaempferol and quercetin were able to accept D-glucose thoughn less efficiently. Consequently, the possibility of sequential steps of 3-O- and then 7-O-glucosylations of flavonols to give the 3,7-di-O-glucoside was discussed.

Synthesis of carotenoids is photoregulated in many fungi including Neurospora crassa. In order to investigate the regulatory mechanism at the enzyme level, several carotenoid mutants of Neurospora were used to determine the activities of enzymes involved in the carotenoid bio synthetic pathway after growth under illumination or in darkness. Light stimulation of carotenoid formation was due to enhanced activities of three subsequent enzymes, geranylgeranyl pyrophosphate synthase, phytoene synthase, and phytoene desaturase indicating a coordinated regulation at the enzyme level. Farnesyl pyrophosphate synthase and lycopene cyclase were not involved in light regulation. Immunological studies showed that in the case of phytoene desaturase higher activity in the light originated from an increased amount of this enzyme in light-grown cultures.

Natural polysaccharides were examined for their activity as elicitors of phytoalexins and flavonoids in alfalfa cotyledons. Only two polysaccharides, laminaran and pectic acid, had elicitor activity. Laminaran, which was more active than pectic acid, was hydrolyzed by tunicase and the hydrolysate was subjected to charcoal and gel filtration columns. Introduction of the pyridylamino group into the elicitor-active oligosaccharides was attempted in order to facilitate isolation. The pyridylamino derivatives were found to exhibit higher activity than the original oligosaccharides. Their liquid chromatography mass spectrometry (LC-MS) analysis revealed that the elicitor-active principles form two ion clusters with the same molecular weights, m/z 1070 and 1232. Their high performance liquid chromatography (HPLC) analysis showed three main peaks. The individual peaks (LN-1,2,3) were collected and subjected to the alfalfa cotyledon assay. LN-3 showed the highest activity (minimum effective concentration, 0.8 μg/ml).

Proteins that react with anti-hum an spectrin antibodies raised in rabbit were found in pea seedlings and leaves. The immunoreactive proteins seem to be associated with the membranes and can be extracted with low ionic strength solutions.

During greening of pigment mutant C-2A′ of Scenedesmus obliquus the iron tetrapyrrole protoheme is synthesized via 5-aminolevulinic acid (ALA). In this organism ALA can be formed either via the C 5 - or the Shemin-pathw ay. [ 14 C]labeling with precursors of both pathways led to radioactive protoheme. [1- 14 C]glutamate, the substrate of the C 5 -pathway, labeled protoheme 7 times more efficiently than [2- 14 C ]glycine, a substrate most effective for the Shemin-pathway. Labeling experiments in the presence of different inhibitors were carried out to prove the specificity of the observed labeling. Gabaculine, a highly effective inhibitor of glutamate-1-semialdehyde aminotransferase, one of the enzymes of the C 5 -pathway, led to a decrease in [1- 14 C ]glutamate incorporation, and to an increase in [2- 14 C]glycine label, showing that the pathways work independently from each other. With DCMU only the incorporation of [1- 14 C ]glutamate was reduced, indicating that the C 5 -pathway activity is depending on plastidic energy supply. The presented data confirm the existence of both pathways to 5-aminolevulinic acid in the pigment mutant C-2A′ of Scenedesmus and their involvement in protoheme formation. It is concluded that the C 5 -pathway provides the bulk of protoheme for the plastidic electron transport chain and the Shemin-pathway for the minor part of the hemes needed for the electron transport in the mitochondria.

In the pest control of stored products like grain and flour, treatment with gases like CO 2 or nitrogen plays an increasing role, as this type of treatment leaves no residues in the food product and is less questionable than treatment with ionizing radiation. An important pest of stored grain is the beetle Tribolium confusum. T. confusum pupae, youn g and aged adults were exposed to pure nitrogen, CO 2 or air at different temperatures between 38 and 46 °C. In nitrogen as well as in CO 2 pupae were the most resistant. Nitrogen efficacy on adult mortality was much higher than CO 2 which was higher than air. For pupae CO 2 was more efficient than nitrogen or air in the range 38 - 45 °C. The temperature dependence of the sensitivity to both gases was noticeable in every stage, particularly below a given specific temperature. It was concluded that the combination controlled-atmosphere-temperature could be a valuable time saving and non aggressive technique for the stored-foods insects control.

The recent identification of the fusicoccin-binding protein (FCBP) in plasma membranes from monocotyledonous and dicotyledonous angiosperms has opened the basis for an elucidation of the toxin’s mechanism(s) of action and indicated a widespread occurrence of the FCBP in plants. Results of a detailed taxonomic survey of fusicoccin-binding sites are reported. Binding sites were not found in prokaryotes, animal tissues, fungi and algae including the most direct extant ancestors of the land plants (Coleochaete). From the Psilotales (Psilophytatae) to the monocotyledonous angiosperms, all taxa analyzed possessed high-affinity microsomal fusicoccin-binding sites. A heterogeneous picture emerged for the Bryophvta. Anthoceros crispulus (Anthocerotae), the only hornwort available to study, lacked fusicoccin binding. Within the Hepaticae as well as the Musci, species lacking and species exhibiting toxin binding were found. The binding site thus seems to have emerged very early in the evolution of the land plants. The tissue distribution of fusicoccin-binding sites was studied in Vicia faba L. shoots. All tissues analyzed showed fusicoccin binding, although not to the same extent. On a per-cell basis, guard cells were found to contain, compared to mesophyll cells, a nine-fold higher number of binding sites. Based on cell surface area, the site density is by a factor of 32 higher in guard cells than in mesophyll cells. Tissue specific expression of the binding sites is suggested by these findings.

Departement de Botanique, Service de Photobiologie, Universite de Liege, Sart-Tilman, Belgium and Georg H. Schmid

In vitro translated proteins from poly(A + )RNA of immature barley starchy endosperm and embryos were immunoadsorbed with antibodies raised against proteins of aleurone layers, starchy endosperm and embryos. Four starchy endosperm and eight embryo specific transcripts were detected. In addition, several mRNA s were restricted to only two of the three tis­sues. Comparing SDS-PAGE patterns of the in vivo protein extracts against which the antibodies were raised, four aleurone, six starchy endosperm and four embryo-specific protein bands were detectable. As for the in vitro translated proteins, several in vivo protein bands were here present in only two of the three tissues. Of eight known barley grain proteins for which antibodies were available, only three were present in developing embryos.

The colorless embryos of Cuphea wrightii A. Gray accumulate capric (about 30%) and lauric acid (about 50%) in their storage lipids. Fractionation studies show that the capacities for the synthesis of these medium-chain fatty acids (MCFA) from [1- 14 C]acetate were strictly bound to intact plastids. These, in turn, obligately required the addition of ATP. ATP could partially be substituted by ADP. Reduction of the pyridine nucleotide pool, required for opti­mum MCFA formation within the plastids, was driven by glucose 6-phosphate. Under these conditions the plastids were capable of synthesizing MCFA like the intact tissue. The presence of CoA in the incubation medium induced acyl-CoA formation. The observed accumulation of unesterified capric and lauric acid in the absence of CoA suggests that acyl-ACP thioesterase activity is involved in the chain termination. Treatment with cerulenin led to an unexpectedly small reduction of total fatty acid synthesis while the chain elongation of capric acid was clearly inhibited. A similar accumulation of capric acid at the expense of longer chain fatty acids has been observed after replacing ATP by ADP. These findings implicate that even the condensing enzymes are involved in the control of chain ter­mination.

Determination of binding of antibodies to lipids onto the surface of the thylakoid mem­ brane, before and after the removal of the CF 1 -complex with sodium bromide, showed that in the immediate vicinity of CF 1 sulfolipid and monogalactolipid occur in higher concentration and are therefore arranged in domains. The m olar ratio of the CF 1 -complex to glycolipids was determined in Nicotiana tabacum chloroplasts of different structure. Thus, in the chlorophyll-deficient tobacco mutants N. tabacum Su/su and Su/su var. Aurea, the molar ratio of CF 1 / monogalactolipid is the same and found to be 1:570. The structure of the lamellar system in these mutants is characterized by a higher ratio of stroma lamellae to grana stacks when compared to the green wild type. In the wild type the ratio CF 1 /monogalactolipid is 30 per cent larger (1 :740). In contrast to this the m olar ratio CF 1 /sulfolipid and CF 1 /digalactolipid is the same in the wild type and the Su/su mutant, whereas these ratios are twice as high in the yellow mutant Nicotiana tabacum Su/su var. Aurea. The binding of glycolipids and phospholipids onto the subunits of CF 1 from Spinacia olera-cea was determined in the Western blot procedure by using monospecific antisera. These experiments lead to the result that the two large subunits (α and β) are marked by antisera to monogalactosyldiglyceride, digalactosyldiglyceride and sulfoquinovosyldiglyceride. The antisera to phospholipids react differently: whereas the antiserum to phosphatidylinositol only reacts with the a-subunit, the antiserum to phosphatidylcholine and that to phosphatidylglycerol react just as the antisera to glycolipids with both large subunits. It is observed that the antiserum to monogalactolipid occasionally marks the y-subunit. This might mean that the glycolipids and the respective phospholipids are tightly bound onto the reacting α-and β-subunits of the CF 1 -complex. Incubation of the subunit CF 1 with lipase from Rhizopus arrhizus and with phospholipase C from Chlostridium perfringens after their transfer to the nitrocellulose membrane abolishes the positive reaction of the peptides with the antisera to glycolipids and phospholipids.

This study deals with the structural perturbations that the aminoglycoside antibiotic gentamicin (GENT) can produce to phospholipid bilayers. Two multibilayer systems, one built-up of dimyristoylphosphatidylcholine (DMPC) and the other of dimyristoylphosphatidylethanolamine (DMPE) were allowed to interact with GENT. The experiments were performed in both a hydrophobic and a hydrophilic medium below the phospholipid main transition tem­peratures. X-ray diffraction techniques were used to determine the extent of the perturbation induced by GENT. The maximum effect was attained when GENT interacted with DMPC in the hydrophobic medium. On the other hand, GENT in aqueous solutions was unable to per­turb in any significant extent the structure of the phospholipids under study.

Analysis of the marine sponge Verongia aerophoba from the Canary islands afforded the brominated secondary constituents isofistularin-3, aerophobin-1 and aerophobin-2 which are probably involved in the chemical defense of the sponge. In addition the yellow pigment uranidine and the unusual sterol aplysterol were isolated. The patterns of brominated compounds were almost superimposable when samples of V. aerophoba from different islands were com­ pared by HPLC indicating de novo synthesis by the sponge or by endosymbiotic microorgan­isms rather than uptake by filter feeding. The only differences observed between the different samples analyzed were with regard to the total concentrations of brominated compounds which varied from 7.2-12.3% of the dry weight dependent on the different collection sites. The Opisthobranch gastropod Tylodina perversa is specialized for feeding on V. aerophoba. Chemical analysis of the gastropod revealed the sponge constituents uranidine, isofistularin-3, aerophobin-1 and aerophobin-2 as well as aerothionin, a further brominated compound which is apparently a biotransform ation product of the brominated sponge constiiuents.

For cytogenetics of pig (Sus scrofa domestica) and the influence of chromosome aberrations on pig production, high interest exists in flow sorted chromosomes for gene mapping, to estab­lish DNA-libraries, or to produce DNA-probes. Flow karyotyping and sorting as well as slit scan flow analysis of metaphase chromosomes of an abnormal cell type carrying a translocation marker chromosome 6/15 are described. Flow sorting of the largest chromosomes of these cells was performed. After sorting the chromosomes still had a well preserved morphology and were identified microscopically by G-banding. The quality of the band pattern of the sorted chromosomes was compatible to that of isolated chromosomes not subjected to flow cytometry. The sorted fraction showed an enrichment of chromosom e 6/15 and chromosome 1 which have quantitatively about the same integrated fluorescence intensity. Slit scan flow analysis was performed to discriminate these two chromosomes. Metacentric and submetacentric chromosom es were analyzed according to their bimodal slit scan profiles. Profiles of the largest chromosomes were distinguished by their different centromeric indices. Two groups were interpreted as the normal chromosome 1 and the translocation chromosom e 6/15.

Dynamics (MD) simulations were performed on a gramicidin A dimer model representing a transmembrane channel. Different from previous simulations the peptide was in contact with bulk water at both ends of the dimer to guarantee a realistic description of the hydration of the biomolecule. The flexible BJH model for water was employed in the simula­tions and the gramicidin-water, gramicidin-ion and ion-water potentials used are based on molecular orbital calculations. The water structure near the gramicidin was investigated first by a simulation without ions, while for the energy profiles of the ion transport through the channel a potassium or a sodium ion was added. These investigations provide a detailed and conclusive picture on a molecular level of the role of water in the ion transport through a gramicidin A channel and can explain the experimental results on the selectivity between alkali ions, their double or even triple occupancy, the exclusion or permeability of anions depending upon cation concentration and the consequences of differences in the ionic charge. The investi­gation demonstrate that the water molecules around the gramicidin behave as an integral part of the peptide and the functionality is the result of the whole complex biomolecule-water.

Nitrogen oxide binding to isolated tomato fruit cuticles has been characterized using Fourier transform infrared spectroscopy. The performed infrared analysis indicates that nitration occurs in the flavonoids of mature tomato cuticles.

The structure of specific granules from horse eosinophil leucocytes is still largely unknown. In this work, electron microscopical studies of horse eosinophils reveal that the large cytoplasmic granules contain an external membrane, a m atrix o f less density, and a dense (non crystalline) core. Round vacuolar inclusions of matrix materials were often observed within the cores. Horse eosinophil granules showed a considerable heterogeneity, and three morphological types could be identified according to structural features of the core and matrix.