Hansjörg A.W. Schneider

Light mediated increase in activity of porphobilinogen deaminase/uroporphyrinogen III cosynthetase and δ-aminolevulinate dehydratase in tissue cultures of tobacco

Abstract Purified enzyme extracts from tissue cultures of tobacco which have been submitted to continuous white illumination show an increase in activity of porphobilinogen deaminase/uroporphyrinogen III cosynthetase and δ-aminolevulinate dehydratase. The specific activities increase linear over a range of about 4 days. Following a lag phase, the chlorophyll content increase is also linear. The time courses show that multiplication of enzymatic activities of the porphyrin biosynthesis chain is involved in chlorophyll synthesis.

On a Blue Light Effect and Phytochrome in the Stimulation of Georesponsiveness of Maize Roots

Summary Maximal stimulation of the geotropic response of maize roots (Badischer Landmais) occurs in red and blue light. A plateau and two peaks at 455 and 484 nm in the blue region and a broad maximum extending from the yellow to the dark red region can be distinguished if the light dose is low (1.3 to 2.6 nE/CM 2 ) and applied during a short period of time. The geostimulation by light can largely be reverted by far red light given after a stimulus. There is no doubt that phytochrome is involvedbut the spectral response curve points toward the participation of additional photoreceptors e.g. the blue light photoreceptor. Responses occuring in yellow light cannot easily be traced back to a known photoreceptor. In the light of existing hypotheses about geotropism and the action and location of morphogenetically active photoreceptors wthe results may best be interpreted by the assumtion that light-stimulated changes in membran permeability may allow a better flow of growth inhibiting substances coming from the root cap.

Highly Efficient Purification of the Labile Plant Enzyme 5-Aminolevulinate Dehydratase (EC 4.2.1.24) by Means of Monoclonal Antibodies

Abstract 5-Aminolevulinate dehydratase (ALAD) from spinach (Spinatia oleracea) was isolated by affinity purification on an immunoabsorbens with a yield of 70 to 80% of the activity in the crude enzyme preparation. The enzyme eluted from the immunoabsorbens was pure as judged by poly acrylamide gel electrophoresis and is a hexamer with a subunit molecular weight of about 50000. Enzyme bound to the immunoabsorbens was able to synthesize porphobilinogen in a continuous manner. Owing to the lability of the enzyme and its low abundance in plant tissue, we have been unable to obtain similar yields of purified enzyme using classical purification procedures. This highly efficient purification was made possible by using monoclonal antibodies as described by Köhler and Milstein (Nature 256, 495 (1975)). The availability of monoclonal antibodies meant that it was not necessary to purify the enzyme to homogeneity by classical means in order to raise an antiserum specific for ALAD. Sixteen clones of cells producing anti bodies against ALAD were selected. They all expressed a ϰ light chain but differed in the heavy chain class which was either γ1 or γ2a. The availability of pure ALAD enzyme and of highly specific antibodies against the enzyme now enables us to answer questions concerning properties, localization, intercellular transport and evolution of ALAD. It is clear that the technique used and the questions asked are not restricted to ALAD.

Determination of the sites of synthesis of chlorophyll synthesizing enzymes in cell cultures of nicotiana tabacum

Abstract The activity of a sequence of enzymes involved in chlorophyll biosynthesis (δ-aminolevulinic acid synthetase (ALAS), δ-aminolevulinic acid dehydratase, porphobilinogenase and chlorophyllase) was followed during greening of tobacco cell cultures under the influence of chloramphenicol (CAP). The photosynthetic enzymes ribulose diphosphate carboxylase (RuDPCO) and NADP linked glyceraldehyde dehydrogenase (NADP-GDH) were used as markers for penetration and action of the inhibitor. RuCPCO was inhibited at concentrations of CAP which still allowed good chlorophyll accumulation. The enzymes of chlorophyll biosynthesis, the activity of which increased during illumination and CAP treatment, behaved like NADP-GDH which is known to be synthesized in the cytoplasm. The results suggest that synthesis of enzymes of chlorophyll biosynthesis takes place in the cytoplasm. Decreasing light induced increment of ALAS activity caused by CAP may possibly be taken as an indication that things are more complicated with this enzyme.

Light-mediated growth inhibition of maize roots

Summary Growth inhibition of maize roots can be evoked by a short pulse of light. The inhibition is scarcely overcome before 10 to 12 hours. Under continuous illumination the threshold for growth inhibition was less than 2.5 · 10 −10 mol quanta · m −2 s −1 . Red light seems to be most effective, but green light of fluence rates far below those of green «safe light» still exerts a strong influence on root growth. Red/far-red photoreversibility was not observed.

δ-Aminolävulinatsynthetase, δ-Aminolävulinatanreicherung und Chlorophyllsynthese in Zellkulturen von Tabak

Summary The experiments are part of a more comprehensive work about porphyrin and chlorophyll synthesis in greening plants. δ-Aminolevulinate synthetase, the enzyme limiting protochlorophyllid synthesis in etiolated leaves of corn, is shown to be active in dark grown tissue cultures of tobacco. When the cultures are illuminated and become green, the activity of δ-aminolevulinate synthetase increases as well as the activity of δ-aminolevulinate dehydratase and porphobilinogenase, whereas the activity of succinyl CoA synthetase is scarcely affected by light. No protochlorophyllid is found in dark grown cells. δ-Aminolevulinate added to the cultures inhibits chlorophyll synthesis and growth, when the concentrations in the cells are higher than 5 · 10 −6 mole/l. The inhibition by δ-aminolevulinate is very specific; a comparable inhibition by levulinate is only observed with concentrations more than a hundred fold higher. The present results are very different from those reported with corn leaves. Knowing that the ability to synthetize chlorophyll is inevitably linked with the development of the plastids, the differences are attributed to the different types of plastids in maize and tissue cultures of tobacco.

Properties of Lectins from Snails of the Genus Helix Probed by Monoclonal Antibodies

Abstract Helix-Lectins, Monoclonal Antibodies against Lectins, C om petition between Antibodies and Carbohydrates Monoclonal antibodies were raised against the lectin of H elix pom atia (HPL). Besides anti bodies bearing the more common y and x chains, antibodies with a, /j. and X2 chains were elicited. The anti-HPL antibodies are expected to be useful in studies on HPL biogenesis and HPL sub structure and in studies concerned with the binding of HPL to cell surfaces. Binding of carbohydrates to HPL im paired the binding of anti-H PL antibodies. One to 3 m M GalNAc inhibited HPL-binding in two out of nine antibodies. None of the antibodies bound in the presence of micrograms per ml of the polyvalent blood group A-substance from hog stomach. Similarly, all anti-HPL antibodies were prevented from binding if non-inhibitory concentrations of A-substance were supplem ented with GalNAc. Lectins from H elix aspersa (HAL) and H elix lucorum (H LL) differed from HPL in antigenic properties. Only one anti-HPL antibody each bound these lectins as well as HPL. Binding of lectins of Cepaea and Rapana was scarcely detectable. Most of the anti-HPL antibodies and the m ultivalent H PL-antigens formed precipitation lines in double diffusion tests. At least two antibodies (IgMs) did so with HLL but none with HAL. The possibility that antibodies were selected because of unknown interactions between HPL and the carbohydrate moieties of certain fractions of antibodies was excluded by raising the antibodies in the presence of tunicamycin to inhibit N-glycosylation. Monoclonal antibodies were raised against the lectin of Helix pomatia (HPL). Besides antibodies bearing the more common γ and ϰ chains, antibodies with α, μ and λ2 chains were elicited. The anti-HPL antibodies are expected to be useful in studies on HPL biogenesis and HPL substructure and in studies concerned with the binding of HPL to cell surfaces. Binding of carbohydrates to HPL impaired the binding of anti-HPL antibodies. One to 3 mᴍ GalNAc inhibited HPL-binding in two out of nine antibodies. None of the antibodies bound in the presence of micrograms per ml of the polyvalent blood group A-substance from hog stomach. Similarly, all anti-HPL antibodies were prevented from binding if non-inhibitory concentrations of A-substance were supplemented with GalNAc. Lectins from Helix aspersa (HAL) and Helix lucorum (HLL) differed from HPL in antigenic properties. Only one anti-HPL antibody each bound these lectins as well as HPL. Binding of lectins of Cepaea and Rapana was scarcely detectable. Most of the anti-HPL antibodies and the multivalent HPL-antigens formed precipitation lines in double diffusion tests. At least two antibodies (IgMs) did so with HLL but none with HAL. The possibility that antibodies were selected because of unknown interactions between HPL and the carbohydrate moieties of certain fractions of antibodies was excluded by raising the antibodies in the presence of tunicamycin to inhibit N-glycosylation.