Mikuláš Pšenák

Biochemical aspects of the biosynthesis of opium alkaloids

Abstract Enzyme systems contained in poppy plants ( Papaver somniferum L.) at different stages of development catalyse hydroxylations, oxidative deaminations, transaminations and decarboxylations of precursors of opium alkaloids. Activities of corresponding enzymes were demonstrated by both in vivo and in vitro experiments. Hydroxylation of phenylalanine could not be demonstrated, but an active phenolase was present catalysing the hydroxylation of tyrosine and the oxidation of 3,4-dihydroxyphenylalanine. These reactions were inhibited by sodium diethyldithiocarbamate and activated by l -ascorbic acid. A direct relation was observed between phenoloxidase activity and the content of the main opium alkaloids (thebaine, codeine, morphine) in poppy plants. Deamination of different amino acids, including aromatic amino acids, was demonstrated by in vitro experiments in the presence ofcatechol: corresponding oxo-derivatives were formed. These substances may also arise in P. somniferum plants by transaminations as was shown by in vitro experiments in the presence of α-ketoglutaric acid and pyridoxal phosphate. Aromatic amino acids were also decarboxylated by enzyme systems of P. somniferum , in these reactions corresponding amines were formed. The results described showed the importance of enzymic catalysis in the transformation of basic precursors in the first phase of biosynthesis of opium alkaloids where the 1-benzylisoquinoline structure is formed.

Biochemical and Physiological Aspects of Developmental Cycle of Colchicum autumnale L.

This study was conducted to examine the individual developmental stages of Colchicum autumnale. We identified the sclerenchymatic tissue in the middle part of the protuberance. This tissue supports the function of protuberance as a kind of hollow diverticulum. On the boundary of the new corm and the shoot a meristematic layer was recognized. We assume that this abscission zone-like structure can initiate dying back of the above-ground part regularly at the end of annual life-cycle. The major part of starch is reutilized in the mother corm during the autumnal stage, supporting sprouting which takes place in the soil. Decline of starch content is paralleled by increasing of total amylolytic activity. From amylolytic enzymes α-amylase, β-amylase and α-glucosidase have been identified. The presence of pullulanase and starch phosphorylase was not observed. From free sugars glucose, fructose and sucrose were identified in corms. The level of sucrose increased significantly during winter season.

Transaminierung und desaminierung von phenylalanin in wurzeln von Zea mays

Zusammenfassung In zellfreien Extrakten aus drei Tage alten Wurzeln von Zea mays konnten die Transaminase und Ammonium-Lyase des l -Phenylalanins durch Fraktionierung mit Ammoniumsulfat bzw. Sephadex G-200-Saulen getrennt werden. Es wurden das pH-Optimum und die Temperatur-Stabilitat dieser Enzyme, sowie die Substratspezifitat der Transaminase bestimmt.

Protein reutilisation in corms of Colchicum autumnale

Colchicum autumnale L. is a monocotyledonous geophyte with hysteranthous leaves, i.e. flowering and leaf growth occur in different time periods. Because after the starch, the second prominent storage compound of corm is represented by proteins, we were interested in nitrogen remobilisation during the annual life cycle of C. autumnale. In this context the content of soluble and insoluble proteins were measured in parallel with determination of some exo-and endopeptidase activities. Our results indicate that the continual proteolysis occurs in both mother and new daughter corms during the whole life-cycle of the plant. L-Ala-aminopeptidase and trypsin-like endopeptidase were the most active peptidases in both mother and daughter corms. As the protein level of mother corm did not change significantly during the development of the future above-ground part under the soil surface (the first, autumnal developmental stage), the developmental profile of nitrate reductase activity was estimated followed by evaluation of total nitrogen and amino acid contents. Significant activity of root nitrate reductase was detected in the roots only in the second, vernal stage. Our results showed that the stored proteins constituted a relevant nitrogen source partly required by the growth processes of the late autumnal stage, but mainly by the intensive growth of leaves and reproductive structures during the second, photosynthetically active stage of the life-cycle.

Glucose transferase in roots of Geum urbanum

Abstract Acetone powders from roots Geum urbanum plants catalysed the intramolecular transfer of glucose; the phenolic glucosides salicin and arbutin were transformed into mono-, di- and probably tri- and tetraglucosides of saligenin and oligoglucoside of quinol respectively. These derivatives were formed without any energy-rich donor of glucose being added to the system. Among newly formed products gentiobioside of saligenin and quinol were identified. The mechanism of isosalicin and saligenin gentiobioside formation is discussed.

Antilipoxygenase activity of compounds from Hypericum perforatum

The main biologically active constituents of Hypericum species are flavonoids (quercetin, isoquercitrin, hyperoside, rutin), biflavonoids and naphthodianthrones (hypericin, pseudohypericin). Lipoxygenase is the key enzyme in the biosynthesis of leukotriens, which have been postulated to play an important role in the pathophysiology of several inflammatory and allergic diseases. This work deals with the investigation of potential antilipoxygenase activity of different compounds and extracts isolated from Hypericum perforatum L. The highest inhibitory effect was exhibited by flavonoid derivative hyperoside (IC50 5.768 × 10−6 M). Acetone and ethanolic extracts caused also an inhibition of lipoxygenase. On the basis of inhibitory effect of compounds tested we assume that the most of them may be involved in the antiinflammatory principles of Hypericum perforatum L.

Characterization of Polyphenol Oxidase from the Latex of Opium Poppy

Polyphenol oxidase from the latex of opium poppy was purified to the electrophoretic homogeneity by affinity chromatography using p-aminobenzoic acid as a ligand coupled to Sepharose CL-4B by divinyl sulphone activation method. The purified enzyme was used to prepare the polyclonal antibodies. The purified latex PPO exhibited high diphenolase activity in comparison with almost unmeassurable monophenolase activity. Both of these activities were sensitive to the activation with sodium dodecyl sulphate. Two isoforms (65 and 40 kDa) of latex PPO were separated by the gel filtration. There were no differences in substrate specifity (weak monophenolase and high diphenolase activity) and sensitivity to inhibitors between these isoforms, but they showed differences in electrophoretic mobility.

The Incorporation of Protoporphyrin IX into Chlorophyll a in Higher Plants and Iron Effects

Summary Using a crude homogenate from tobacco leaves and 14C-protoporphyrin IX as substrate, a stimulatory effect with an iron concentration (up to 2 mM) on 14C-protoporphyrin incorporation into chlorophyll a was observed. When 14C-protoporphyrin monomethyl ester was incubated with homogenates from normal tobacco leaves, no incorporation into chlorophyll was detected.

Phenylalanine Ammonia-Lyase in Poppy Plants (Papaver somniferum L.)1)

Summary The enzyme phenylalanine ammonia-lyase (EC 4.3.1.5.) has been found in seedlings of Papaver somniferum L. var. Vahovecký, isolated and purified by ammonium sulphate precipitation, Sephadex G-100 filtration and a sorption on hydroxylapatite. The enzyme preparation with specific activity approximately 60 times higher than the original homogenate was used for determination of pH optimum, heat stability, apparent Michaelis constant and activation energy. The effects of various hydroxyderivatives of cinnamic acid on this enzyme are also described.

Catalase activity in developing seedling of opium poppyPapaver somniferum L

Catalase is an enzyme unique to glyoxysomes in developing poppy seedlings. Catalase activity is very low in endosperm and in embryo of germinating poppy seeds. During postgerminative growth and development the enzyme activity increases rapidly with maximum in endosperm on day 2 and in developing seedling on day 3. A rapid decline of enzyme activity parallells the extension growth of poppy seedlings. Three electrophoretic forms of catalase have been detected in isolated glyoxysomes and partially purified catalase preparation. Electron microscopic observation indicates the presence of catalase in glyoxysomes of parenchyma cells of poppy seedling cotyledons. Numerous lipid bodies and electron-dense deposits in vacuoles are the most characteristic feature of these cells.