Ion Effects on the H+-Translocating Adenosine Triphosphatase and Pyrophosphatase Associated with the Tonoplast of Chora corallina

H⁺-translocating ATPase and pyrophosphatase (PPase) associatedwith the tonoplast of Chara corallina were isolated with theaid of a perfusion technique, and the effects of ions on theiractivities were studied. All the alkali metal cations testedstimulated the ATPase and ATPdependent H⁺ pumping activitiesonly by 10 to 40%. Anions, on the other hand, strongly affectedthe activities. Potassium salts of Cl^- and Br^- stimulated them,while F^- and NO₃^- inhibited them. By contrast, the H⁺-translocatingPPase was insensitive to anions but sensitive to cations. Theorder of cation stimulation was Rb⁺=K⁺>Cs⁺>Na⁺=Li⁺>choline⁺.NO₃^- (50 mil), thought to be a specific inhibitor of the tonoplast-typeH⁺-ATPase, inhibited the ATPdependent H⁺ pumping almost completelybut the ATPase activity by only about 50%. Na⁺ inhibited thePP₁-dependent H⁺ pumping (I_5O=5OmM) in the presence of 50 mMKCl but not the ATP-dependent one. The PPase was more sensitiveto F^- (I₅₀=400µM) than the ATPase. Both the H⁺-ATPaseand the H⁺-PPase required Mg²⁺ for their activities, althoughan excess was inhibitory to both. The different sensitivitiesof the PP₁-dependent and the ATP-dependent H⁺- pumping enzymesto ions correspond to the tonoplast enzymes of higher plantsand may be used as "markers" to distinguish between these enzymesin characean cells (Received October 2, 1987; Accepted May 18, 1988)     

Biosensor system for continuous flow determination of enzyme activities. II. Simultaneous determination of plural enzyme activities.

A biosensor system for continuous flow determination of plural enzyme activities was prepared from the combination of two pyruvate sensors, a prereactor and a flow cell. This system was applied to the simultaneous determination of lactic dehydrogenase (LDH) and glutamic-pyruvic transaminase (GPT) activities in the same sample. These enzyme activities can be determined by measuring pyruvate produced by the enzyme reactions as follows. The amount of pyruvic acid can also be determined from the amount of oxygen consumed upon oxidation of pyruvic acid by pyruvate oxidase. (Formula: see text). Therefore, both of the detectors for the determination of lactic dehydrogenase and glutamic-pyruvic transaminase activities were prepared from the combination of a pyruvate oxidase membrane and an oxygen electrode. Pyruvate oxidase was covalently immobilized on a membrane prepared from cellulose triacetate. A linear relation was obtained between the output current and LDH or GPT activities in the range of 50 to 3,600 IU l-1 or 6 to 1,000 IU l-1, respectively. Each assay of these enzyme activities was completed within 15 min. The results obtained had a precision of ca. 4%. The sensor was stable for more than 25 days at 5 degrees C.     

Isolation and Identification of α-(γ-Aminobutyryl)-Hypusine

A new dipeptide, alpha-(gamma-aminobutyryl)-hypusine, was identified in bovine brain. This compound was isolated from trichloroacetic acid-soluble fraction of bovine brain with five steps of ion-exchange chromatography. Its structure was postulated by routine chemical analyses and determined by synthesis. The amount of the compound isolated from 1.2 kg of bovine brain was 870 nmol.     

Auxin-Induced Changes in the Cell Wall Xyloglucans: Effects of Auxin on the Two Different Subtractions of Xyloglucans in the Epicotyl Cell Wall of Vigna angularis

In order to study the IAA-induced modifications of the cellwall of azuki bean (Vigna angularis Ohwi et Ohashi cv. Takara)epicotyl segments, the xyloglucans were subfractionated intotwo components, i.e., 4K-U and 24K xyloglucans, which were obtainedby extraction with 4% KOH solution containing 8 M urea and 24%KOH solution, respectively. The weight-average molecular weightsof 4K-U and 24K xyloglucans were estimated to be 40 x 10⁴ and106 x 10⁴, respectively. Complete acid hydrolysis of 4K-U and24K xyloglucans gave glucose, xylose, galactose and fucose inmole % 48.3 : 33.5 : 13.8 : 4.4 and 45.3 : 30.9 : 19.6 : 4.3,respectively. Treatment of epicotyl segments with IAA (0.1 mM) caused a decreasein the amount of 24K xyloglucans and an increase in 4K-U xyloglucans,whereas the total amount of the two xyloglucans remained constant.Furthermore, IAA treatment caused a decrease in the molecularweight of 24K xyloglucans from 106 x 10⁴ to 78 x 10⁴ withoutcausing changes in their sugar compositions. With 4K-U xyloglucans,IAA caused an increase in the mole % of xylose and a decreasein the mole % of galactose and fucose. ¹ This paper is dedicated to the late Professor Joji Ashida. (Received November 26, 1982; Accepted February 7, 1983)     

Effects of vanillin and o-vanillin on induction of DNA-repair networks: modulation of mutagenesis in Escherichia coli

Vanillin and its isomer o-vanillin have an effect on the adaptive and SOS responses, as well as mutagenesis, induced in Escherichia coli by N-methyl-N-nitrosourea (MNU) and UV irradiation, potentiating in some cases and suppressing in others. o-Vanillin markedly inhibited the MNU-induced adaptive response, while both vanillins potentiated the UV-induced SOS response. These phenomena appear to be responsible for the comutagenic or antimutagenic role of these chemicals in MNU and UV mutagenesis.     

Interaction of Benzoquinones with QA- and QB- in Oxygen-Evolving Photosystem II Particles from the Thermophilic Cyanobacterium Synechococcus elongatus

The interactions of benzoquinones with the reduced forms ofthe bound plastoquinone acceptors, Q_A and Q_B, were studied withoxygen-evolving photosystem II (PS II) particles from the thermophiliccyanobacterium Synechococcus elongatus, which largely lack poolplastoquinone molecules [Takahashi and Katoh (1986) Biochim.Biophys. Acta 845: 183]. Oxygen evolution in the presence ofvarious electron acceptors was determined and flash-inducedchanges in absorbance in the blue region were analyzed in termsof difference spectra, dependence on the concentration of benzoquinoneand on temperature, and sensitivity to 3-(3,4-dichlorophenyl)-1,1-dimethylurea(DCMU). The more hydrophobic the quinone molecule, the higherwas the rate of oxygen evolution, and the maximum rate of 3,000µmoles O₂.(mg chlorophyll)^–1.h^–1 was recordedin the presence of phenyl- and dichloro-p-benzoquinones. DCMUinhibited oxygen evolution by more than 95%. However, spectrophotometricstudies revealed that, even though electrons were transferredto benzoquinones predominantly via the direct oxidation of by added benzoquinones occurred in such a way as to indicate thatabout 40% of PS II reaction centers were not associated withfunctional Q_B sites. was very stable in the presence of ferricyanide. However, benzoquinonesinduced the slow oxidation of . The characteristics of the benzoquinone reductioin in thePS II preparation is discussed. ¹Present address: Department of Life Sciences, Faculty of Science,Himeji Institute of Technology, Shosha 2167, Himejishi, Hyogo-ken,671-22 Japan (Received May 8, 1990; Accepted August 14, 1990)     

Mechanism of Electron Flow through the QB Site in Photosystem II. 4. Reaction Mechanism of Plastoquinone Derivatives at the QB Site in Spinach Photosystem II Membrane Fragments

Interactions of externally added plastoquinone (PQ) derivatives(PQ₀-PQ₃) with the photosystem II (PSII) acceptor side wereinvestigated in PSII membrane fragments prepared from spinachby measuring the photoreduction rates of PQ derivatives at variousPQ concentrations, and the following results were obtained. From the kinetic analysis, all the PQ derivatives (PQ₀-PQ₃)except PQ₃ were shown to accept electrons at two sites (theQ_B site and the PQ site) as in the case of Synechococcus vulcanusPSII particles with benzoquinone derivatives [Satoh et al. (1995)Plant Cell Physiol. 36: 597]. Affinities of PQ derivatives at the Q_B site increased as thelength of the isoprene side chain got longer, while those atthe PQ site were not very much different for all the PQ derivativestested in this study. The inhibitory effect of DCMU was noncompetitive, and, therefore,the affinity of PQ₃ for the PQ site was determined while thatfor the Q_B site could not be estimated presumably due to itsfairly high affinity to the site. Based on the results obtained using PQ derivatives, the mechanismof interaction of an authentic PQ, PQ₉, at the Q_B site is discussed. (Received May 2, 1996; Accepted July 24, 1996)     

Poly(ADP-ribose) polymerase: Structural conservation among different classes of animals and its implications

Poly(ADP-ribose) polymerase cDNAs have been isolated from different classes of animals. Cloning of genes from lower eukaryotes has allowed us to investigate directly the biological functions of poly(ADP-ribosyl)ationin vivo. The conservation of specific regions among mammals, chicken,Xenopus laevis, andDrosophila melanogaster reveals the essential structural elements required for recognition of breaks in DNA and for catalytic activity. Cys, His and basic residues in the zinc-finger consensus region are conserved. The carboxyl terminal region corresponding to an NAD-binding domain is strongly conserved. The dinucleotide-binding consensus sequence and 1-A-2, Rossmann fold structure, and -sheet structures are completely conserved from mammals to insect. InDrosophila, a putative leucine-zipper motif has been identified, and other poly(ADP-ribose) polymerases also contain an -helical, amphipathic structure in the auto-modification domain. In this article, we review the recent structural analyses of the functional domains of poly(ADP-ribose) polymerase in phylogenetically divergent species, and discuss the implications of structural conservation for its biological functions.Abbreviations aa amino acid(s) - D. melanogaster Drosophila melanogaster - PARP poly(ADP-ribose) polymerase [EC 2.4.2.30] - PCR polymerase chain reaction - X. laevis Xenopus laevis     

Light-Induced CREB Phosphorylation and Gene Expression in Rat Retinal Cells

Abstract: The signal pathway for light-induced expression of c- fos and the neuropeptide somatostatin (SS) in rat retinal cells was investigated. Flashing light induced c- fos and SS mRNA in the inner nuclear layer and the ganglion cell layer. As both c- fos and SS genes have a cyclic AMP response element (CRE) in their promoters, CRE-binding protein (CREB) phosphorylation in retinal cells was examined with a phospho-CREB-specific antibody. Both flashing light and administration of the L-type Ca²⁺ channel activator Bay K 8644 induced phosphorylation of CREB in the nuclei of the amacrine cells and the ganglion cells where c- fos /SS mRNAs were expressed. These cells could be double-stained with anti-calmodulin kinase II (anti-CaM kinase II) monoclonal antibody and phospho-CREB-specific polyclonal antiserum after Bay K 8644 administration, indicating the colocalization of phosphorylated CREB at Ser¹³³ and CaM kinase II in the neural retina.     

Putative morphogen, DIF, of Dictyostelium discoideum induces apoptosis in rat pancreatic AR42J cells

We have recently shown that differentiation-inducing factor-1 (DIF-1) of Dictyostelium discoideum is capable of raising intracellular calcium concentration ([Ca²⁺]_i) and suppressing cell proliferation of rat pancreatic AR42J cells in a dose-dependent manner, and that DIF-1 at a concentration of 40 μmol/L is toxic to the cells. In this study, we have further characterized the cytotoxic effect of DIF-1 on AR42J cells and have analyzed the effect of DIF-1 on [Ca²⁺]_i. In the presence of 40 μmol/L DIF-1, cells began to bleb after approximately 6 h, and most had died within 48 h. Biochemical analysis revealed that DNA fragmentation was accompanied by cell death. Monitoring the changes in [Ca²⁺]_i induced by DIF-1, it was found that cells were able to adapt to stimulation with DIF-1 so that they did not respond to subsequent stimulation by DIF-1. These results indicate that DIF-1 induced apoptosis in AR42J cells probably via a cell signaling system.