Auslösung von Elementarprozessen durch einzelne Lichtquanten im Fliegenauge

Summary Light quanta impinging upon the photopigments located in the rhabdomeric receptor structures of the fly's compound eyes trigger photochemical reactions which in turn elicit miniature receptor potentials (bumps). The paper mainly deals with the problem whether a single quantum of light is sufficient, or whether a coincidence of quanta and/or elementary photochemical events is necessary to trigger a miniature receptor potential.The experiments were based on tests of the optomotor responses of fixed flying flies suspended in a rotating patterned cylinder with periodic distributions of inner surface brightness. The tests were made under two different light programs: 1) Illumination constant in time 2) Illumination by periodic light pulse sequences with various frequencies. Average light fluxes absorbed by the receptors were equal in both programs. Theoretical considerations lead to the following conclusions: The strength of the optomotor responses to the light programs 1 and 2 should not differ from each other in the case of single quantum processes. However for multiquantum processes light program 2 should be more effective than light program 1 as it favours the coincidence of quantum absorptions per unit time. But these theoretical conclusions are valid only if two conditions are fulfilled in the experiments: a) The pulse frequency of light program 2 has to be kept below a certain limit which is determined by the kinetics of the photochemical systems. Otherwise light program 2 gets averaged in time and in principle can be not more effective than light program 1. b) The rates of quanta absorbed by the receptors have to be kept low enough to guarantee that the concentration of unbleached pigment molecules remains practically unchanged as compared with the concentration in darkness. Accordingly the test experiments were carried out with light pulse frequencies ranging from 500 to 1/120 cycles per second. Intensities were used which corresponded to an average quantum flux effective for one rhabdomeric structure ranging between 10 and 250 quanta per second.The interpretation of the experimental results is in accordance with the hypothesis that one single quantum of light is sufficient to trigger an elementary photochemical reaction and that in turn one single photochemical event can elicit a miniature receptor potential. At present time the experiments do not allow conclusions about the possible occurrence of coincidence-functions of synapses at the level of the first optical ganglion which receive their information via fibers leading off from the receptors.In one of the appendices of the paper, the transinformation flux into a receptor is calculated, taking into consideration the Poisson noise of the quanta disrupting the signal at extremely low quantum rates.

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Ein Teil der in dieser Arbeit abgedruckten Ergebnisse wurde bereits in zwei vorläufigen Mitteilungen publiziert, Reichardt (1965, 1966).     

Evaluation of a mutation test using S49 mouse lymphoma cells and monitoring simultaneously the induction of dexamethasone resistance, 6-thioguanine resistance and ouabain resistance

A test for the detection of chemically induced mutants in S49 mouse lymphoma cells is described. These cells can be plated in parallel in several selective media; the induced frequencies of dexamethasone-resistant, 6-thioguanine-resistant and ouabain-resistant mutants were compared. The first two selection systems permit the detection of all kinds of mutation that result in alteration or partial or complete loss of the gene product concerned, whereas ouabain-resistant mutants can only be induced with strong point mutagens in these cells. Dexamethasone resistance is the marker induced at the highest frequency among these three. Data obtained from this selection system are therefore the most amenable to statistical analysis. Dexamethasone resistance is expressed within a short time after mutagenesis (3 days), and because S49 cells do not display metabolic co-operation, large numbers of cells can be screened. A metabolizing system in vitro with rat-liver homogenate may be included in tests of indirectly acting mutagens. These features make the S49 mutation test system using dexamethasone resistance as the main marker and other markers as internal controls an attractive tool in mutation testing in somatic cells in vitro.     

Bryophytes with asexual reproduction — Their ability to succeed in an industrial area

Summary The extent to which the distribution of liverwort and moss species in the industrial area around Duisburg (Federal Republic of Germany) is influenced by asexual reproduction is studied using distribution data. The research shows that neither the liverworts nor the mosses — except the genus Plagiothecium — are more successful in the area of concentrated industry when they are capable of asexual reproduction.     

Protein Synthesis in BHK-21 Cells Infected with Semliki Forest Virus

[³H]leucine-labeled proteins synthesized in BHK-21 cells infected with Semliki Forest virus were fractionated by polyacrylamide gel electrophoresis (PAGE). Cellular and virus-specific proteins were identified by difference analysis of the PAGE profiles. The specific activity of intracellular [³H]leucine was determined. Two alterations of protein synthesis, which develop with different time courses, were discerned. (i) In infected cultures an inhibition of overall protein synthesis to about 25% of the protein synthesis in mock-infected cultures develops between about 1 and 4 h postinfection (p.i.). (ii) The relative amount of virus-specific polypeptides versus cellular polypeptides increases after infection. About 80% of the proteins synthesized at 4 h p.i. are cellular proteins. Since significant amounts of nontranslocating ribosomes in polyribosomes were not detected up to 7 h p.i., the inhibition of protein synthesis is not caused by inactivation of about 75% of all polyribosomes but by a decreased protein synthetic activity of the majority of polyribosomes. Indirect evidence indicates that an inhibition of elongation and/or release of protein synthesis develops in infected cells, which is sufficient to account for the observed inhibition of protein synthesis. Inhibition of over-all protein synthesis developed when virus-specific RNA began to accumulate at the maximal rate. This relationship was observed during virus multiplication at 37, 30, and 25 C. A possible mechanism by which synthesis of virus-specific RNA in the cytoplasm could inhibit cellular protein synthesis is discussed. Indirect evidence and analysis of polyribosomal RNA show that the increased synthesis of virus-specific protein is brought about by a substitution of cellular by viral mRNA in the polyribosomes.     

Limited proteolysis of yeast phosphofructokinase by subtilisin. Alterations in enzyme activity, subunit composition, and hydrodynamic properties.

Yeast phosphofructokinase having a molecular weight of 750000--800000 (20 S) has been subjected to limited proteolysis by subtilisin and yeast proteases. Two steps of proteolytic degradation could be distinguished: in the first step, which is accompanied by an increase in molecular activity, the subunits alpha and beta (Mr 120000) are converted to alpha' and beta' (Mr approximately 900000), and in the second step, accompanied by a decrease in enzyme activity, alpha' is converted to alpha' (Mr 80000) and two further fragments having Mr 45000 and 35000 become detectable. In the course of the conversion the sedimentation value of the undissociated enzyme drops from 20 S to about 17 S. The two substrates fructose 6-phosphate and ATP exhibit characteristic protective effects on enzyme activity and on subunit degradation. Whereas the first step is not strongly influenced by the substrates, fructose, 6-phosphate inhibits significantly the degradation of alpha' and beta', whereas ATP prevents only degradation of beta'. When in presence of ATP alpha' is degraded to alpha', the quaternary structure of the 17-S enzyme is no longer stable and a dissociation of the molecule occurs to a 12-S form which is enzymically active and ATP-sensitive and in which the ratio of alpha' to beta' is one-to-one.     

Elektrophoretische Untersuchungen über die Wirkung von Gewebe-Fixierungsmitteln auf Eiweisslösungen

Zusammenfassung Die Wirkung verschiedener Gewebe-Fixierungsmittel auf Humanalbumin und die Proteine des Seretins^® (menschliche Serumkonserve) wurde papierelektrophoretisch untersucht. A. Humanalbumin. 1. In nur wenigen Fällen kommt es als Primärwirkung der Fixierungslösungen zu einer Präzipitatbildung. Für alle untersuchten Fixierungsmittel gilt, daß der größere Teil des eingesetzten Humanalbumins nach kurzem Einwirken der Fixierungslösungen als natives Eiweiß wiedergefunden wird. Nur geringe Anteile des Humanalbumins werden in wenigen Fällen durch das betreffende Fixierungsmittel denaturiert. 2. Fünfstündige Einwirkungsdauer von 5% TCE in 6% igem Formol auf das Humanalbumin hat keine verstärkte Wirkung zur Folge. 3. Die Wirkung von 6% igem Glutardialdehyd auf das Humanalbumin ist streng zeitabhängig. Bereits 10 min nach Zugabe des Glutardialdehyds zum Humanalbumin enthält die Lösung kein natives Eiweiß mehr. B. Seretin ^® . 1. Nach ihrer Wirkung auf das Seretin^® lassen sich die untersuchten Fixierungsmittel in drei Gruppen gliedern: a) Fixierungsmittel mit sehr geringer Wirkung. Die Eiweiße des Seretins^® werden durch diese Fixierungsmittel nicht oder kaum vermindert. Nur geringe Eiweißmengen werden denaturiert. Diese Fixierungsmittel wirken auch bei fortschreitender Einwirkungszeit nicht stärker. Zu der Gruppe gehören: Methanol 96%, Osmiumtetroxyd 2% und Formol 10%. b) Fixierungsmittel mit geringer bis stärkerer Wirkung. Meist kommt es nach Zusatz dieser Mittel zur Präzipitatbildung. Teilweise enthalten die Niederschläge aber noch beträchtliche Mengen nativen Eiweißes. Natives Protein wird in jedem Fall gefunden. Denaturiertes Eiweiß liegt in mittleren bis größeren Mengen vor. Die Wirkung dieser Fixierungsmittel ist nur wenig zeitabhängig. Geordnet nach steigender Wirksamkeit handelt es sich um folgende Mittel: TCE 10%, Äthanol 96%, 0,5% TCE in 6% igem Formol, Gemisch nach Carnoy, Perchlorsäure 2%, Gemisch nach Bouin und 5% TCE in 6% igem Formol, c) Stark wirkende Fixierungsmittel. Stets Präzipitat-oder Gelbildung. Weder die Überstände noch die Niederschläge enthalten nach dreistündiger Versuchsdauer noch natives Eiweiß. Die Proteine sind quantitativ denaturiert. Die Wirkung dieser Mittel verstärkt sich mit fortschreitender Einwirkungsdauer. Es handelt sich um die Lösung Susa nach Heidenhain und das Gemisch nach Stieve sowie um Glutardialdehyd 2% und 6%. — 2. Bei der Einwirkung von 0,5% TCE in 6% igem Formol auf das Seretin^® konnte zwischen 0 und 37° C keine Temperaturabhängigkeit der Wirkung gefunden werden. — 3. 5% TCE in 6% igem Formol wirkt mit steigender Temperatur zunehmend stärker denaturierend auf die Seretineiweiße ein. Während bei einer Versuchstemperatur von 0° C nach 3 Std noch größere Mengen nativen Eiweißes wiedergefunden wurden, enthielt die Lösung bei einer Versuchstemperatur von 37° C nach dieser Zeit nur noch denaturiertes Eiweiß. Es scheint hier aber auch schon zu einer Eiweißhydrolyse zu kommen.

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Summary The influence of various tissue fixatives on human albumin and the proteins of Seretin^® (bottled human serum) is tested by means of electrophoresis. A. Human Albumin. 1. Only in a few cases the formation of a precipitate is the first reaction to a treatment with fixatives. All fixatives tested show, that if the treatment lasts for only a short period, the major part of the human albumin treated with fixative is recovered as native protein. In a few cases a small quantity of the human albumin is denatured by the respective fixative. — 2. Treating human albumin with 5% trichloracetic acid (TCE) in 6% formalin for 5 hours shows no increased effect. — 3. The action of 6% glutardialdehyde on human albumin is strictly time-dependent. 10 minutes after glutardialdehyde is added to human albumin no native protein is demonstrable in the solution. B. Seretin ^® . 1. According to their action on Seretin the fixatives in question can be divided in three groups: a) Fixatives which have very little effect. The proteins of Seretin^® are little or not at all affected by the action of the fixative. Only very small quantities of protein are denatured. The effect of these fixatives does not increase with increasing time. The group includes: Methanol 96%, osmiumtetroxyd 2%, and formalin 10%. b) Fixatives with slight to stronger effects. In most cases a precipitate will form after the fixative has been added to the protein solution. Some of the precipitates, however, contain considerable amounts of native protein. Native protein is demonstrable in any case. Denatured protein is demonstrable in medium to large amounts. The effect of these fixatives is hardly influenced by time. In order of increasing effect the fixatives in question are: TCE 10%, ethanol 96%, 0,5% TCE in 6% formalin, Carnoy's solution, perchloric acid 2%, Bouin's solution, and 5% TCE in 6 % formalin. c) Fixatives with strong effects. They regularly induce the formation of either a precipitate or a gel. After three hours neither supernates nor precipitates contain any native protein. The proteins are qualitatively denatured. The effect of the fixatives increases with the time of treatment. The fixatives in question are: Susa solution (Heidenhain), Stieve's solution, glutardialdehyde 2% and 6%. — 2. Temperatures between 0° C and 37° C have no influence on the effect of 0,5 % TCE in 6% formalin on Seretin. — 3. With rising temperatures 5% TCE in 6% formalin has an increasing denaturing effect on the proteins of Seretin^®. While larger quantities of native protein are found after 3 hours a t0° C, only denatured proteins are demonstrable after the same period of time at 37°. It seems, however, that in this case too a protein hydrolysis takes place.

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Abkürzungen H A Humanalbumin - TCE Trichloressigsäure Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.Wir danken Herrn P. Sillmann für seine technische Unterstützung.     

A sialic acid-binding lectin from ovine placenta: purification,specificity and interaction with actin

A sialic-acid-specific lectin from ovine placental cotyledons was purified by affinity chromatography on bovine submaxillary mucin-agarose followed by gel filtration, and it showed a molecular weight of 65 000 by sodium dodecylsulfate-polyacrylamide gel electrophoresis. This lectin has the capacity to interact with actin, since in binds to actin-F in a cosedimentation assay and it acts as a mediator in the binding of action to the affinity column. The lectin agglutinated rabbit and rat erythrocytes, but not human A, B or O erythrocytes. Haemagglutination inhibition assays of different saccharides, glycoproteins and glycolipids indicate that this lectin has affinity for sialic acid, which is enhanced by its O-acetylation. The N-terminal sequence of the protein shows 92% identity with rabbit and porcine uterine calreticulin.     

Competition between β-ketothiolase and citrate synthase during poly(β-hydroxybutyrate) synthesis inMethylobacterium rhodesianum

The enzymes -ketothiolase and citrate synthase from the facultatively methylotrophicMethylobacterium rhodesianum MB 126, which uses the serine pathway, were purified and characterized. The -ketothiolase had a relatively highK _m for acetyl-CoA (0.5 mM) and was strongly inhibited by CoA (K _i 0.02 mM). The citrate synthase had a much higher affinity for acetyl-CoA (K _m 0.07 mM) and was significantly inhibited by NADH (K _i 0.15 mM). The intracellular concentration of CoA metabolites and nucleotides was determined inM. rhodesianum MB 126 during growth on methanol. The level of CoA decreased from about 0.6 nmol (mg dry mass)^-1 during growth to the detection limit when poly(-hydroxybutyrate) (PHB) accumulated. Nearly unchanged intracellular concentrations of NADH, NADPH, and acetyl-CoA of about 0.5, 0.6–0.7, and 1.0 nmol (mg dry mass)^-1, respectively, were determined during growth and PHB synthesis. During growth, the -ketothiolase was almost completely inhibited by CoA, and acetyl-CoA was principally consumed by the citrate synthase. During PHB accumulation, the -ketothiolase had about 75% of its maximum activity and showed much higher activity than citrate synthase, which at the actual NADH concentration was about 75% inhibited. NADPH concentration was sufficiently high to allow the unlimited activity of acetoacetyl-CoA reductase (K _mNADPH 18 M). PHB synthesis is probably mainly controlled by the CoA concentration inM. rhodesianum MB 126.Abbreviation PHB Poly(-hydroxybutyrate)     

Glutamine synthetase oligomers and isoforms in sugarbeet (Beta vulgaris L.)

The -amino-N compounds that accumulate in the thickening storage root of sugarbeet (Beta vulgaris L.) were synthesized in the leaves (NO ₃ ^– nutrition) and also in the lateral roots (NH ₄ ⁺ nutrition). Ammonium stimulated glutamine synthetase (GS, EC 6.3.1.2) activity, especially in the lateral roots. With non-denaturing polyacrylamide-gel isoelectric focussing, simultaneously active charge-isomers of GS were separated in both leaves and roots. The leaf isoforms were active in an octameric and also in a tetrameric form. In the root only octameric isoforms were found. The tetramer was more active than the octamer in the leaf blade and vice versa in the leaf stem. Only the tetramer needed -mercaptoethanol for activity stabilization in vitro. A reactivation, however, of an inactive tetramer by the addition of thiol/thioredoxin was not possible. The same isoforms of GS were separated in different organs of sugarbeet but with different patterns of relative activity. The activity pattern depended also on the N-source of the plant. With increasing age of the plant the number of active GS isoforms declined in both leaves and roots although the in-vitro activity remained unchanged (NO ₃ ^– -fed plants) or even increased (NH ₄ ⁺ -fed plants).Abbreviations GS glutamine synthetase (E.C. 6.3.1.2.) - IEF isoelectric focussing - PAGE polyacrylamide gel electrophoresis This work was supported by a grant from Bundesministerium für Forschung und Technologie and by Kleinwanzlebener Saatzucht AG, Einbeck.