Characterization of synchronized cultures of Bumilleriopsis filiformis

Activity of the photosynthetic apparatus of synchronized cultures was studied with the xanthophycean alga Bumilleriopsis filiformis, following the kinetics of fluorescence induction and photooxidation of cytochrome f (= cytochrome c-553) of intact cells. During the beginning of the cell-division phase, minimum cellular photosynthetic activity is observed and a maximum after its completion, which is accompanied by corresponding changes in Hill reaction activity and re-reduction of cytochrome f by photosystem II light. At minimum activity, the level of steady state fluorescence was higher than at the maximum. This is due, at least in part, to the diminished electron flow between the two photosystems seemingly caused by decreased photosystem I activity. This explanation was suported by the kinetics of cytochrome-f photooxidation.Thus, electron transport activity of both photosystems appears to vary during the cell cycle.Abbreviations pBQ p-benzoquinone - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DCIP dichlorophenolindophenol - MV methylviologen (paraquat) - Q fluorescence quencher (in photosystem II)     

Electron microscopy of plastids of Bumilleriopsis filiformis

The chloroplast structure of Bumilleriopsis filiformis shows typical characteristics reported for algae and particularly Xanthophyceae. Three thylakoids are often grouped forming lamellae, but the apposed membranes do not fuse; patterns involving more than three thylakoids also occur. A girdle lamella-present in plastids of different algae groups and also considered to be typical for Xanthophyceae-is absent; starch, and distinctive pyrenoids could not be observed either. Besides the chloroplast envelope the plastid is surrounded by and ER cisterna which partly forms a tubular periplastidal reticulum between the two encircling double membranes.     

Herbicide resistance in a mutant of the microalga Bumilleriopsis filiformis

A DCMU* (diuron)-resistant algal mutant was selected and characterized. Chlorophyll content, growth, and photosystem-I activity are as in the wild-type. Growth in liquid medium with 3 M DCMU present is half of the control. Apparently only the herbicide-binding site is affected within the redox chain. In contrast to the wild-type, trypsin treatment of isolated chloroplast material completely abolishes photosynthetic electron transport inhibition by DCMU or atrazine.DCMU resistance of chloroplasts is accompanies by tolerance to triazinones and phenylpyridazinones, but not to symmetric triazines. Sensitivity to diphenylethers, DBMIB or o-phenanthroline is not altered.Data on this algal mutant combined with those from triazine-resistant mutants of higher plants give direct evidence of overlapping binding sites at a (hypothetical) binding protein located at the reducing side of photosytem II.     

Isolation and Characterization of Microbodies from the Alga Chlorogonium elongatum

The alga Chlorogonium elongatum was grown autotrophically or heterotrophically on acetate. Cells harvested in the logarithmic phase of growth were disrupted, and the whole homogenates were fractionated on sucrose gradients. Protein and enzyme determinations carried out on the fractions led to the following conclusions. Chloroplast fragments which represent the major portion of particulate protein in autotrophic cells migrate to density 1.17 g/cm³. In heterotrophic cells, mitochondria comprise most of the particulate protein, and these particles accumulate at density 1.19 g/cm³, as shown by a peak of cytochrome oxidase in this region. Part of the catalase and uricase, two marker enzymes for microbodies, were found in the soluble fractions, but 60% or more of these activities were recovered at density 1.225 g/cm³ from autotrophic cells. Electron micrographs showed that in this region there were microbodies with a diameter of 0.4 micrometer. The isolated microbodies contained no isocitrate lyase, a marker enzyme of glyoxysomes. This enzyme was completely soluble and therefore seems not to be associated with organelles in this organism.     

Isolation and Biochemical Characterization of Peroxisomes from Cultured Rat Glial Cells

Peroxisomes are now recognized to play important cellular functions and its dysfunction leads to a group of neurological disorders. This study reports peroxisomal enzyme activities in cultured glial cells and peroxisomes isolated from cultured oligodendrocytes and C₆ glial cells. Peroxisomal enzyme activities were found to be higher in oligodendroglial cells than in astrocytes or mixed glial cells. We also developed a method for the isolation of peroxisomes from glial cells by a combination of differential and density gradient centrifugation techniques. Peroxisomes from oligodendrocytes in nycodenz gradient were isolated at a density of 1.165 g/ml ± 0.011. Activities of dihydroxyacetone phosphate acyl transferase, -oxidation of lignoceric acid and -oxidation of phytanic acid were almost exclusively associated with the distribution of catalase activity (a marker enzyme for peroxisomes) in the gradient. This protocol should be a resource for studies designed to investigate the structure and function of peroxisomes in brain cells.     

Amino acid sequence of a ferredoxin from Bumilleriopsis filiformis, a yellow-green alga: relationship with red algae, protoflorideophyceae, and filamentous blue-green algae

The amino acid sequence of a [2Fe-2S] ferredoxin isolated from Bumilleriopsis filiformis, a yellow-green alga, was determined by using conventional techniques. It consisted of 98 amino acid residues with a microheterogeneity at the amino-terminus: Ala/Glu-Thr-Tyr-Ser-Val-Thr-Leu-Val-Asn-Glu-Glu-Lys-Asn-Ile-Asn-Ala-Val- Ile- Lys-Cys-Pro-Asp-Asp-Gln-Phe-Ile-Leu-Asp-Ala-Ala-Glu-Glu-Gln-Gly-Ile-Glu- Leu- Pro-Tyr-Ser-Cys-Arg-Ala-Gly-Ala-Cys-Ser-Thr-Cys-Ala-Gly-Lys-Val-Leu-Ser- Gly- Thr-Ile-Asp-Gln-Ser-Glu-Gln-Ser-Phe-Leu-Asp-Asp-Asp-Gln-Met-Gly-Ala-Gly- Phe- Leu-Leu-Thr-Cys-Val-Ala-Tyr-Pro-Thr-Ser-Asp-Cys-Lys-Val-Gln-Thr-His-Ala- Glu- Asp-Asp-Leu-Tyr. No prominent structural feature was noted in this ferredoxin in comparison with other homologous ferredoxins. From the structural comparison, B. filiformis was placed taxonomically close to filamentous blue-green algae and red algae.     

Peroxisomes in the Alga Vaucheria are Neither of the Leaf Peroxisomal Nor of the Glyoxysomal Type*,1

Microbodies were isolated from the freshwater alga Vaucheria sessilis as well as from a marine Vaucheria. The organelles equilibrated on sucrose gradients at densities 1.23 g . cm^?3 and 1.24g . cm^?3, respectively. On electron micrographs they showed an ovoid or spheroid shape with a diameter of 0.5 to 0.8 μm. Besides catalase, the peroxisomes of both algae possess glycolate oxidase and glutamate-glyoxylate aminotransferase, but no other leaf-peroxisomal enzymes. Instead, the enzymes malate synthase and isocitrate lyase, which are markers of glyoxysomes in higher plants, are constituents of the peroxisomes in the marine as well as in the freshwater alga. Citrate synthase, aconitase, malate dehydrogenase and enzymes of the fatty acid β-oxidation pathway are located exclusively in the mitochondria. Therefore, the peroxisomes from Vaucheria do not belong to either the type of leaf peroxisomes or to the type of glyoxysomes.     

Myeloperoxidase from Neutrophil Peroxisomes

Peroxisomal myeloperoxidase plays a key role in synthesis of oxidants by neutrophilic leukocytes. This heme protein consists of two subunits connected by a disulfide bond. The enzyme uses ₂₂ and l^– for synthesizing HOCl, the major oxidant produced by neutrophils. In addition to the chlorination reaction, myeloperoxidase exhibits some other properties depending on its oxidation state. The enzyme significantly affects synthesis of oxidants in the cells depending on the competing substrate concentrations and other factors. ^¯ ₂ is also a physiological substrate of myeloperoxidase. Its reaction with the enzyme determines how the cells utilize ^¯ ₂ for pathogen elimination. ^¯ ₂ affects the chlorinating and peroxidase activities of myeloperoxidase. In addition, ^¯ ₂ reacts with the enzyme yielding the catalytically active compound III that hydroxylates phenols.     

Purification and Characterization of Cadmium-Binding Protein from Unicelluar Alga Chlorella sorokinian

The unicellular green alga Chlorella sorokiniana ANA9 is highly resistant to heavy metals, and its metal-binding proteins are induced in the presence of cadmium. A novel cadmium-binding protein in C. sorokiniana cultured in 100 mg/l cadmium ions for 4 days was isolated and characterized. The crude protein extract was obtained by cell disruption and partly purified by ammonium sulfate precipitation. After purification by anion-exchange chromatography with diethylaminoethyl (DEAE)-Sepharose CL-6B, the protein was further purified by gel filtration with Sephacryl S-100, followed by Sephadex G-75. The molecular weight of the purified protein was determined to be 11.5 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The cadmium binding capacity of the purified protein was 119 μg/mg. The involvement of thiol coordination in metal-ion binding was confirmed by measuring the ultraviolet spectrum. This article is the first to describe the metallothionein-like cadmium-binding protein from Chlorella species, the expression of which is induced by cadmium exposure.     

Characterization of a Highly Efficient Blue-shifted Channelrhodopsin from the Marine Alga Platymonas subcordiformis

Rhodopsin photosensors of phototactic algae act as light-gated cation channels when expressed in animal cells. These proteins (channelrhodopsins) are extensively used for millisecond scale photocontrol of cellular functions (optogenetics). We report characterization of PsChR, one of the phototaxis receptors in the alga Platymonas (Tetraselmis) subcordiformis. PsChR exhibited ∼3-fold higher unitary conductance and greater relative permeability for Na⁺ ions, as compared with the most frequently used channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2). Photocurrents generated by PsChR in HEK293 cells showed lesser inactivation and faster peak recovery than those by CrChR2. Their maximal spectral sensitivity was at 445 nm, making PsChR the most blue-shifted channelrhodopsin so far identified. The λ_max of detergent-purified PsChR was 437 nm at neutral pH and exhibited red shifts (pK_a values at 6.6 and 3.8) upon acidification. The purified pigment undergoes a photocycle with a prominent red-shifted intermediate whose formation and decay kinetics match the kinetics of channel opening and closing. The rise and decay of an M-like intermediate prior to formation of this putative conductive state were faster than in CrChR2. PsChR mediated sufficient light-induced membrane depolarization in cultured hippocampal neurons to trigger reliable repetitive spiking at the upper threshold frequency of the neurons. At low frequencies spiking probability decreases less with PsChR than with CrChR2 because of the faster recovery of the former. Its blue-shifted absorption enables optogenetics at wavelengths even below 400 nm. A combination of characteristics makes PsChR important for further research on structure-function relationships in ChRs and potentially useful for optogenetics, especially for combinatorial applications when short wavelength excitation is required.     

Purification and Characterization of a New Pigment from the Green Alga, Bryopsis maxima

Several, new, water-soluble pigments have been detected in thematured thalli of the green alga, Bryopsis maxima. Among thepigments, a major red one has been purified and characterized.The red pigment has absorption maxima at 237, 268, 331, 450,485 and 520 nm and a shoulder at 570 nm. Its fluorescence emissionspectrum has maxima at 659 and 730 nm. The pigment has minuscharge at the pH above 3.0 and is soluble in water and polarorganic solvents but not in nonpolar solvents. Its molecularweight was estimated to be 1,490. The infrared, N.m.r. and massspectra suggest that the pigment has an open tetra pyrrole structure. ⁵Present address: Department of Biochemistry, Nippon MedicalSchool, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113, Japan. (Received July 2, 1987; Accepted August 26, 1987)     

大鼠肝细胞过氧化物酶体的提取

:采用蔗糖密度梯度离心法 ( 950 0 0× g,2 h)提取大鼠肝细胞过氧化物酶体 ,所得过氧化物酶体形态完整 ,纯度与肝匀浆相比提高了 2 6倍 ,仅有少量 ( 0 .5%～ 0 .9%)的微粒体和线粒体污染 ,回收率为 1 2 %。为研究过氧化物酶体提供了有效的分离方法。此法还可将过氧化物酶体、微粒体、线粒体同时进行分离。     

Characterization of Sulfate Transport in the Green Alga Chlorella ellipsoidea

A rapid induction of sulfate transport was observed in the greenalga Chlorella ellipsoidea during sulfur-limited growth. Bothaffinity and V_max increased about five-fold within 6 h of transferringcells from Bold's basal medium with 350 µM MgSO₄ to sulfur-deficientBold's medium. High affinity sulfate transport was induced within15 min and reached maximum rate within 3 h of transferring cellsto sulfur-deficient condition, indicating that a new, high-affinity-sulfatetransport system is induced by sulfur starvation in C. ellipsoidea.Eadie-Hofstee plots of initial rates of sulfate uptake indicatedthat the K of sulfur-starved cells was about 17 µM. Bothsulfur-starved and unstarved cells grown in air had a V_max of1.5 times higher than that of high-CO₂ grown cells. Sulfatetransport was completely inhibited by 30 µM CCCP or 800µMKCN both in the light and the dark but transport in the lightwas not inhibited by 20 µM DCMU. Treatment with 50 µMor 500 µM vanadate caused 50% inhibition of uptake. Therate of sulfate uptake in the dark was twice that in the lightand was stimulated by low pH. These results suggest that thesulfate transport system in C. ellipsoidea is operated by protonsymport across the plasmamembrane which is partially mediatedby P-type ATPase and that these systems depend exclusively onenergy derived from oxidative phosphorylation in the mitochondria. (Received June 28, 1995; Accepted August 8, 1995)     

Further Biochemical Characterization of a Cell Fusion Hormone from the Red Alga, Griffithsia pacifica

In the marine red alga, Griffithsia pacifica, the repair processinitiated upon death of an intercalary cell is mediated by aglycoprotein hormone, rhodomorphin. In this paper we show thatthe glycosidic portion of the molecule has terminal -D-mannosylresidues which are at least in part responsible for the bindingof rhodomorphin by concanavalin A. The protein portion of themolecule contains disulfide bridges. These bridges must be intactfor biologically active hormone to be recovered from a denaturedstate. Isolation of active rhodomorphin from SDS-PAGE indicatesa molecular weight of 15,000–17,500. This agrees wellwith our previously published [Watson and Waaland (1983) PlantPhysiol. 71: 327] molecular weight from gel filtration of 14,000. (Received February 26, 1986; Accepted June 2, 1986)     

Isolation and Characterization of the Cytoplasmic Membranes from the Blue-Green Alga (Cyanobacterium) Anacystis nidulans

Cells of the blue-green alga (cyanobacterium) Anacyslis nidulanswere disintegrated, and their thylakoid membranes and cytoplasmicmembranes were isolated by floatation centrifugation on a sucrosedensity gradient. Electron micrographs revealed that the cytoplasmicmembranes formed single closed vesicles having diameters of200–400 nm. These membranes contained xanthophylls asthe major constituent pigments and rß-carotene andchlorophyll a as very minor ones. The major peaks in their absorptionspectra were due to carotenoids at 435, 455 and 487 nm, witha minor one due to chlorophyll a at 673 nm. These findings areconsistent with the yellow color of the cytoplasmic membranes.The absorption spectrum of the membranes in the carotenoid regionwas markedly affected by temperature: with a decrease in temperature,the peaks at 455 and 487 nm diminished and a new peak appearedat 390 nm. (Received February 12, 1983; Accepted June 20, 1983)     

Purification and Molecular and Immunological Characterization of a Unique Phosphoribulokinase from the Green Alga Selenastrum minutum

A unique phosphoribulokinase (ADP:D-ribulose 5-phosphate 1-phosphotransferase, EC 2.7.1.19) has been purified to homogeneity from the green alga Selenastrum minutum. The enzyme has a native molecular mass of about 83 kilodaltons and a native isoelectric point of 5.1. The enzyme consists of two different-sized subunits of 41 and 40 kilodaltons, implying that it is a heterodimer. This is the first report of a eukaryotic heterodimeric phosphoribulokinase. The in vivo existence of two nonidentical subunits of S. minutum phosphoribulokinase was confirmed by western blot analysis of crude protein extracts from trichloroacetic acid-killed cells. These two subunits were immunologically similar, as rabbit immunoglobulin G affinity purified against the 41 kilodalton subunit of S. minutum phosphoribulokinase (PRK) cross-reacts with the 40 kilodalton subunit and vice versa. Antibodies against S. minutum phosphoribulokinase also cross-react with the spinach enzyme. NH₂-terminal sequencing revealed that the two S. minutum PRK subunits shared a considerable degree of structure homology with each other and with the enzymes from spinach and Chlamydomonas reinhardtii, but not with PRK from Rhodobacter sphaeroides. There are, however, differences between the NH₂-terminal amino acid sequences of the two S. minutum PRK subunits, that imply that they are the products of separate genes or products of two different mRNAs spliced from a single gene.