Chromatic Regulation of Photosystem Composition in the Cyanobacterial Photosynthetic System: Kinetic Relationship between Change of Photosystem Composition and Cell Proliferation

Kinetics of the change of photosystem (PS) composition in cyanobacteriainduced by chromatic light were studied in relation to cellproliferation. The study was made for two unicellular strains,Synechococcus NIBB 1059 and Synechocystis (Aphanocapsa) PCC6714. We found that (1) the change to a higher or lower PS I/IIratio was due to acceleration or suppression of apparent PSI formation, and (2) it progressed on a similar time scale tothat of the cell proliferation. The apparent rate constant ofthe change in the PS I/II ratio was proportional to that ofcell proliferation, µ, when this was low, but at highvalues of µ the increase in the rate constant of the changein the PS I/II ratio became smaller, causing a deviation fromthe linear relationship. Results indicate that under autotrophicconditions, the photoregulated composition change occurs asa result of thylakoid development, which accompanies cell proliferation. (Received June 23, 1986; Accepted December 5, 1986)     

Nudeotide Pools and Purine Metabolism in Tissue Cultures of Wild-Type Datura innoxia and Adenine-Requiring Auxotroph

Cells of the auxotrophic mutant, Ad1, of Datura innoxia requiredadenine, adenosine, or inosine for their growth on solid agarmedium which contained Murashige-Skoog salts, 2,4-dichloro-phenoxyaceticacid, and sucrose. Thirteen purine and pyrimidine nucleotidesin extracts of wild-type and Ad1 cells were separated and quantifiedby HPLC. Levels of ADP-glucose and UMP were significantly higherin Ad1 than in wild-type cells, but those of other nucleotideswas found when Ad1 cells were transferred to fresh medium withoutadenine. The rate of the biosynthesis de novo of purines, asestimated from the rate of incorporation of ¹⁴C from [2-¹⁴C]-glycine and [¹⁴C]formate into adenine nucleotides, was reducedin Ad1 cells to 21 and 13% of the wild-type rate, respectively.The activities involved in the salvage of adenine and adenosinein Ad1 cells were similar to those in wild-type cells. Ad1 cellshad the capability to convert adenine to guanine nucleotidesand guanine to adenine nucleotides. ¹ Part 27 of the series, "Metabolic Regulation in Plant CellCulture". (Received March 7, 1988; Accepted August 3, 1988)     

THE EFFECT OF IRON NUTRITION ON PHOTOSYNTHESIS AND NITROGEN FIXATION IN CULTURES OF TRICHODESMIUM (CYANOPHYCEAE)1

Cultures of Trichodesmium NIBB 1067 were grown in the synthetic medium AQUIL with a range of iron added from none to 5 × 10^?7 M Fe for 15 days. Chlorophyll-a, cell counts, and total cell volume were two or three times higher in medium with 10^?7 M Fe than with no added Fe. Oxygen production rate per chlorophyll-a was over 60% higher with higher iron. Increased iron stimulated photosynthesis at all irradiances from about 12–250 μE · m^?2· s^?1. Nitrogen fixation rate, estimated from acetylene reduction, for 10^?7 and 10^?8 M Fe cultures was approximately twice that of the cultures with no added Fe. The range of rates of O₂ production and N₂ fixation in cultures at the iron concentrations we used were similar to the rates from natural samples of Trichodesmium from both the Atlantic, and the Pacific oceans. This similarity may allow this clone to be used, with some caution, for future physiological ecology studies. This study demonstrates the importance of iron to photosynthesis and nitrogen fixation and suggests that Trichodesmium plays a central role in the biogeochemical cycles of iron, carbon and nitrogen.     

Photoreversible absorption changes of guanidine-HCltreated phycocyanin and allophycocyanin isolated from the blue-green alga Tolypothrix tenuis

In the search for the photoreceptor in photocontrolled phycoerythrinformation, photoreversible absorption changes of chromoproteinsin vivo and in vitro were studied with the blue-green alga Tolypothrixtenuis. Neither intact cells nor crude extracts of soluble proteinsshowed any significant absorption changes which were reversiblyinduced by green and red light. However, the photoresponse wasobservable when the crude protein extracts were treated withthe chaotropic reagent guanidine-HCl (0.4 M, for 1 hr in thedark). Isolated phycocyanin and allophycocyanin also showedthe same photoresponse after the guanidine-HCl treatment. Thedifference spectrum (green minus red) of guanidine-HCl-treatedphycocyanin was almost identical with that shown by phycochromea of Bj?rn and Bj?rn (3), and the allophycocyanin showed thesame difference spectrum as those of phycochrome c of Bj?rnand Bj?rn and the photoreversible pigment isolated by Scheibe(7). Urea at a concentration higher than 1 M or alkaline incubation(pH 8.5) also showed the same effect. The results were interpretedas indicating that phycocyanin and allophycocyanin obtain theability for photoresponsiveness when their protein conformation,probably around the chromophore site, is modified. (Received October 30, 1978; )     

The Pro to glycine mutation of staphylococcal nuclease simplifies the unfolding—folding kinetics

Kinetics of unfolding and refolding of a staphylococcal nuclease mutant, in which Pro¹¹⁷ is replaced by glycine, have been investigated by stopped-flow circular dichroism, and the results are compared with those for the wild-type protein. In contrast to the biphasic unfolding of the wild-type nuclease, the unfolding of the mutant is represented by a single-phase reaction, indicating that the biphasic unfolding for the wild-type protein is caused by cis-trans isomerization about the prolyl peptide bond in the native state. The proline mutation also simplifies the kinetic refolding. Importance of the results in elucidating the folding mechanism is discussed.     

Comparison of cytochrome P-450 species which catalyze the hydroxylations of the aromatic ring of estradiol and estradiol 17-sulfate

For identification of microsomal cytochrome P-450 (P-450) enzymes which catalyze 2- or 4-hydroxylations of estrogens in the rat liver, estradiol (E₂) and estradiol 17-sulfate (E₂-17-S) were selected as the substrates and incubated with various kinds of purified P-450 enzymes: PB-1, PB-2, PB-4 and PB-5 obtained from phenobarbital-treated male rats (Sprague-Dawley); MC-1 and MC-5 from 3-methylcholanthrene-treated male rats; and UT-1, UT-2, UT-4 and UT-5 from untreated animals. The reactions were carried out under the P-450-reconstructed system, and the resulting products were determined by HPLC using electrochemical detection. All the enzymes tested were shown to have varying degrees of catalytic activities for 2-hydroxylation of the two substrates; UT-1 and UT-2 had the highest activity. Of the induced P-450 enzymes, PB-2 and MC-1 showed fairly high catalytic activity for 4-hydroxylation of E₂. The P-450 enzymes obtained from the untreated male rats, especially UT-4, showed the highest catalytic activity for 4-hydroxylation of the two substrates. From these results and also from kinetic experiments, the P-450 enzymes which catalyze 2- and 4-hydroxylations of estrogen were considered to be different species. A part of E₂ was converted to such metabolites as estrone and those having a hydroxyl group at positions 6β, 15 or 16, each production of which was estimated to be catalyzed by single or multiple P-450s.     

Kinetics of the reconstitution of hemoglobin from semihemoglobins α and β with heme

Kinetics of the reconstitution of hemoglobin from semihemoglobins and with hemin dicyanide have been investigated using three kinds of stopped-flow technique (Soret absorption, fluorescence quenching of tryptophan, and Soret CD). The semihemoglobins and are occupied by heme in the and chains, respectively, the other chain being heme-free. Based on the kinetic results, the following scheme for the reconstitution is proposed; First, hemin dicyanide enters the pocket-like site of the apo chains. Second, in semihemoglobin , the CN-ligand in the fifth coordination position of iron is replaced by the imidazole ring of the proximal His immediately after the heme insertion. In contrast, semihemoglobin changes its conformation after the heme insertion, and this is followed by the ligand replacement. Finally, the partial structure changes induced by the ligand replacement propagate onto the whole molecule and the final conformation is attained. The results indicate that semihemoglobin retains a more rigid and organized structure, and more closely approaches its final structure than does semihemoglobin . Correspondence to: Y. Kawamura-Konishi     

Impact of whitefish on an enclosure ecosystem in a shallow eutrophic lake: changes in nutrient concentrations,phytoplankton and zoobenthos

Large bag-type (75 m³) and tube-type (105 m³) enclosures were set up in the shallow eutrophic Lake Suwa and were each stocked with exotic planktivorous whitefish (Coregonus lavaretus maraena). The release of whitefish caused the increase in nutrient concentration in the tube-type enclosure whereas no such increase was observed in the bag-type enclosure. Bottom sediment seemed to be an important source of chironomid food for whitefish. The proportion of phytoplankton measuring<10μm and 20–40μm, which respectively corresponded toOchromonas spp. andCryptomonas sp., were lower in the fish enclosures than in the control, which might have been caused by high grazing pressure by rotifers. The predation by whitefish might have affected the species composition of phytoplankton through reducing copepod predation on rotifers, not through reducing the densities of cladocerans which directly feed on phytoplankton as many investigators have reported. The phytoplankton biomass was not affected much by the release of fish. Possible reasons are that the increase in density of rotifers reduced the biomass of available phytoplankton and also that inedible Cyanophyceae were in the decreasing phase of their seasonal succession and could not increase successfully in spite of elevated nutrient levels.     

Antiserum to Nitrogenase Generated from an Amplified DNA Fragment from Natural Populations of Trichodesmium spp

A fragment of the nifH gene was amplified from natural populations of Trichodesmium spp. and cloned into a maltose-binding protein (MBP) expression vector. The peptide product of the amplified 359-bp fragment of nifH was cleaved from the fusion protein, purified, and used to generate a specific antibody to the Fe protein of nitrogenase. The antiserum recognized the MBP-nitrogenase fusion protein and the cleaved nif peptide product but not MBP. The antibody cross-reacted with nitrogenase from natural populations of Trichodesmium spp. from the Caribbean Sea and with a cultured isolate from the Kuroshio waters (Trichodesmium sp. strain NIBB1067). The same nifH fragment was amplified, cloned, and sequenced from Trichodesmium sp. strain NIBB1067 and was found to be 98% identical at both the protein and DNA levels to nifH from the Caribbean populations. Three of the six nucleotide differences between the Trichodesmium sp. strain NIBB1067 and the Trichodesmium spp. nifH sequence had also been found in a second sequence from the natural populations, indicating either that there is more than one strain of Trichodesmium sp. in natural assemblages or that there are multiple copies of nifH in the genome. This DNA fragment, which is easily amplified with the polymerase chain reaction, may provide a good indicator of species relatedness without requiring extensive cloning or sequencing. Furthermore, the use of the polymerase chain reaction in combination with a MBP protein fusion vector provides a rapid method for production of highly specific sera, starting with a small amount of DNA.