Purification and characterization of cytochrome c6 from the unicellular green alga Scenedesmus obliquus

Purification of a soluble cytochrome c6 from the unicellular green alga Scenedesmus obliquus by a simple and rapid method is described. The purification procedure includes ammonium sulfate precipitation and non-denaturating PAGE. The N-terminal sequence of the first 20 amino acids was determined and shows 85% similarity and 75% identity to the sequence of cytochrome c6 from the green alga Monoraphidium braunii. The ferrocytochrome shows typical UV/VIS absorption peaks at 552.9, 521.9 and 415.7 nm. The apparent molecular mass was estimated to be 12 kDa by SDS-PAGE. EPR-spectroscopy at 20 K shows resonances indicative for two distinct low-spin heme forms.     

Purification and spectroscopic characterization of a recombinant chloroplastic hemoglobin from the green unicellular alga Chlamydomonas eugametos

Hemoglobins (Hb), which have the important task of delivering molecular oxygen by facilitating its reversible binding to the heme, are now thought to have evolved in all groups of organisms including prokaryotes, fungi, plants and animals. Our recent finding of a light-inducible chloroplastic Hb in the green unicellular alga Chlamydomonas eugametos has further extend this idea, while raising questions about the function that an Hb could play in a high oxygen environment such as in the chloroplast. In order to understand the role played by this new Hb, we have undertaken its biochemical characterization. To facilitate the characterization of Chlamydomonas Hb, which represents less than 0.01% of the soluble protein in the green alga, the protein has been expressed in Escherichia coli and purified to apparent homogeneity. The purified recombinant protein possesses a non-covalently bound iron-protoporphyrin IX heme. The oxy form of the recombinant Hb. purified directly from bacterial cells, is very stable, with a measured half-life of 7 days at pH 8 and has an ultraviolet/visible spectrum similar to those of the related cytoplasmic Hbs of the ciliated protozoa Paramecium and Tetrahymena and of the cyanobacterium Nostoc commune. In contrast to what has been reported for oxymyoglobins and oxyhemoglobins, the dioxygen molecule bound to the L1637 Hb can be reduced by the electron-transfer mediator phenazine methosulfate in the presence of NADPH, indicating that the heme pocket of Chlamydomonas Hb may be more accessible to small molecules. With regard to this we found that when the small reducing agent sodium dithionite is used to reduce the met form, it must be removed anaerobically from the Hb prior to oxygenation of the protein to stably produce the oxy form. Otherwise, the oxy form is obtained readily from the met form under an oxygenic atmosphere when ferredoxin and ferredoxin NADP+ reductase are used to enzymically reduce the Hb. Finally, the spectra of the deoxy and met forms were unusual, the heme being partly low-spin at physiological pH. These results confirm the existence of a reversible oxygen-binding protein in the chloroplast of C. eugametos. The unusual spectral and biochemical properties of the protein may reflect a specialized function for this Hb.     

Solution structure of oxidized cytochrome c6 from the green alga Monoraphidium braunii

Cytochrome c6 from Monoraphidium braunii, an 89-amino acid electron transfer protein, has been investigated by NMR in solution, in its oxidized form, at pH 7 and 300 K. By using a combination of COSY, TOCSY, and NOESY experiments, 84% of the proton resonances have been assigned. A total of 1668 experimental NOE constraints, 1109 of which were meaningful, together with 288 pseudocontact shifts, have been used to determine the structure in solution. This is represented as a family of 40 structures which have been energy minimized. The rmsd values with respect to the mean structure are 0.57 +/- 0.08 and 0.94 +/- 0.09 A for the backbone and heavy atoms, respectively. The structure has been found to be very similar to that of the reduced form, except for a rearrangement in propionate 7, a feature which has been observed in all c-type cytochromes investigated so far. Such a feature could be relevant for the efficiency of the electron transfer pathway with either the oxidizing or the reducing partners. Other differences in the oxidation states have been noted in the region proposed to be involved in the interaction with the physiological partners.     

The 1.5-A crystal structure of plastocyanin from the green alga Chlamydomonas reinhardtii

The crystal structure of plastocyanin from the green alga Chlamydomonas reinhardtii has been determined at 1.5-A resolution with a crystallographic R factor of 16.8%. Plastocyanin is a small (98 amino acids), blue copper-binding protein that catalyzes the transfer of electrons in oxygenic photosynthesis from cytochrome f in the quinol oxidase complex to P700+ in photosystem I. Chlamydomonas reinhardtii plastocyanin is an eight-stranded, antiparallel beta-barrel with a single copper atom coordinated in quasitetrahedral geometry by two imidazole nitrogens (from His-37 and His-87), a cysteine sulfur (from Cys-84), and a methionine sulfur (from Met-92). The molecule contains a region of negative charge surrounding Tyr-83 (the putative distant site of electron transfer) and an exclusively hydrophobic region surrounding His-87; these regions are thought to be involved in the recognition of reaction partners for the purpose of directing electron transfer. Chlamydomonas reinhardtii plastocyanin is similar to the other plastocyanins of known structure, particularly the green algal plastocyanins from Enteromorpha prolifera and Scenedesmus obliquus. A potential "through-bond" path of electron transfer has been identified in the protein that involves the side chain of Tyr-83, the main-chain atoms between residues 83 and 84, the side chain of Cys-84, the copper atom, and the side chain of His-87.     

Studies on the cultivation and utilization of the alga Scenedesmus acutus as a singel cell protein

Growth yields, enzyme activities, cytochrome concentrations and the rates of product formation were determined in Propionibacterium shermanii cultures grown in a chemostat with lactate as the energy source at various concentrations of oxygen. Oxygen was toxic when its partial pressure in the inflowing gas was just sufficient to give measurable dissolved oxygen concentration in the culture, when it inhibited lactate oxidation and NADH oxidase activity. Below this oxygen concentration, P. shermanii behaved as a facultative anaerobe. The adaptation from anaerobic metabolism to aerobic metabolism, however, was complex. Low partial pressures of oxygen led to decreased cytochrome and membrane-bound dehydrogenase activities and molar growth yield. Above an oxygen partial pressure of 42 mmHg in the inflowing gas stream, these changes were reversed, leading to an aerobic type of metabolism. At the highest subtoxic concentration of oxygen used (330 mmHg in the input gas), lactate was oxidized mainly to acetate and carbon dioxide and the rate of propionate formation was very low. The high molar growth yield obtained under these conditions suggested that lactate and NADH oxidation via the cytochrome electron transport system was coupled to ATP synthesis.     

Purification and characterization of oxygen-evolving photosystem II core complexes from the green alga Chlamydomonas reinhardtii

Oxygen-evolving photosystem II complexes were isolated from the green alga Chlamydomonas reinhardtii by selective solubilization of thylakoid membranes with dodecyl maltoside followed by density gradient centrifugation and anion-exchange chromatography. In the presence of CaCl2 and K3[Fe(CN)6] the complexes evolved oxygen at rates exceeding 1000 mumol (mg of chl)-1 h-1. The particles contained 40 chlorophylls a and had properties very similar to those of PSII isolated from higher plants. Chlamydomonas reinhardtii is now the first organism which can be used for both site-directed mutagenesis and detailed biochemical and biophysical characterization of oxygen-evolving photosystem II. It seems therefore to be an ideal model organism for investigation of structure-function relationships in photosynthetic oxygen evolution.     

Molecular characterization of a single mitochondria-associated double-stranded RNA in the green alga Bryopsis

OBJECTIVE: To evaluate the effect of a moderate dose of fish oil on glycemic control and in vivo insulin action in type 2 diabetic men with elevated plasma triacylglycerols and to determine the effect of the same treatment on gene expression of GLUT4, lipoprotein lipase (LPL), and hormone-sensitive lipase (HSL) in the abdominal adipose tissue. RESEARCH DESIGN AND METHODS: A total of 12 type 2 diabetic men were randomly allocated to 2 months of 6 g daily of either fish oil or sunflower oil, separated by a 2-month washout interval, in a double-blind crossover design. RESULTS: For glucose metabolism, 2 months of fish oil supplementation compared with sunflower oil led to similar fasting plasma insulin, glucose, and HbA1c. Basal hepatic glucose production did not increase after fish oil. There was no difference in insulin suppression of hepatic glucose production nor in insulin stimulation of whole-body glucose disposal measured by the euglycemic-hyperinsulinemic clamp. Fish oil did not ameliorate the low mRNA level of GLUT4 in adipose tissue of these patients. For lipid profile, fish oil lowered plasma triacylglycerol more than sunflower oil (P < 0.05) and tended to increase the amount of mRNA of both LPL and HSL in adipose tissue. CONCLUSIONS: A moderate dose of fish oil did not lead to deleterious effects on glycemic control or whole-body insulin sensitivity in type 2 diabetic men, with preserved triacylglycerol-lowering capacities.     

Evidence for independent acquisition of group I introns in green algae

We report the occurrence of a group I intron, 452 nucleotides in length, in the nuclear small-subunit ribosomal gene of the benthic seaweed Urospora penicilliformis, a member of the green algal class Ulvophyceae. Group I introns have been reported in fungi, myxomycetes, the ciliate genus Tetrahymena, and recently in five unicellular chlorophycean algae. The sequence of the conserved core of the U. penicilliformis group I intron was aligned with that of 15 other introns of this type, and the evolutionary relationships among these introns were examined. Comparison of the "intron" tree with phylogenetic hypotheses of the intron-harboring organisms shows that nuclear group I introns in green algae have arisen several times and are not lineage specific.     

Complete nucleotide sequence of the chloroplast genome from the green alga Chlorella vulgaris: the existence of genes possibly involved in chloroplast division

The complete nucleotide sequence of the chloroplast genome (150,613 bp) from the unicellular green alga Chlorella vulgaris C-27 has been determined. The genome contains no large inverted repeat and has one copy of rRNA gene cluster consisting of 16S, 23S, and 5S rRNA genes. It contains 31 tRNA genes, of which the tRNALeu(GAG) gene has not been found in land plant chloroplast DNAs analyzed so far. Sixty-nine protein genes and eight ORFs conserved with those found in land plant chloroplasts have also been found. The most striking is the existence of two adjacent genes homologous to bacterial genes involved in cell division, minD and minE, which are arranged in the same order in Escherichia coli. This finding suggests that the mechanism of chloroplast division is similar to bacterial division. Other than minD and minE homologues, genes encoding ribosomal proteins L5, L12, L19, and S9 (rpl5, rpl12, rpl19, and rps9); a chlorophyll biosynthesis Mg chelating subunit (chlI); and elongation factor EF-Tu (tufA), which have not been reported from land plant chloroplast DNAs, are present in this genome. However, many of the new chloroplast genes recently found in red and brown algae have not been found in C. vulgaris. Furthermore, this algal species possesses two long ORFs related to ycf1 and ycf2 that are exclusively found in land plants. These observations suggest that C. vulgaris is closer to land plants than to red and brown algae.     

The sex-inducing pheromone and wounding trigger the same set of genes in the multicellular green alga Volvox

The sex-inducing pheromone of the multicellular green alga Volvox carteri is a glycoprotein that triggers development of males and females at a concentration <10(-16) M. By differential screening of a cDNA library, two novel genes were identified that are transcribed under the control of this pheromone. Unexpectedly, one gene product was characterized as a lysozyme/chitinase, and the other gene product was shown to encode a polypeptide with a striking modular composition. This polypeptide has a cysteine protease domain separated by an extensin-like module from three repeats of a chitin binding domain. In higher plants, similar protein families are known to play an important role in defense against fungi. Indeed, we found that the same set of genes triggered by the sexual pheromone was also inducible in V. carteri by wounding.     

Sulfoquinovosyl diacylglycerols from the alga Heterosigma carterae

An extract of the chloromonad Heterosigma carterae (Raphidophyceae), cultivated in natural seawater, contained a complex mixture of sulfoquinovosyl diacylglycerols. Palmitoyl (16:0), three isomers of hexadecenoyl (16:1 cis delta 9, delta 11, delta 13), and eicosapentenoyl (20:5) were found to be the main fatty acyl substituents. Exact double-bond sites were determined by mass spectrometry analysis of the corresponding nicotinyl derivatives. Four major sulfoquinovosyl diacylglycerol components were partially purified and identified as 1-4 by interpretation of their nuclear magnetic resonance and mass spectral data. In addition, complete analysis of the H. carterae sulfoquinovosyl diacylglycerols was performed using high-performance liquid chromatography combined with electrospray tandem mass spectrometry.     

Evidence for soluble and nuclear site I binding of estrogens in human aorta

The purpose of this study was to establish the estrogen receptor (ER) expression and content in human aorta fragments removed at the time of by-pass surgery. To this end, we adopted a radioligand binding assay to evaluate either soluble (S) or nuclear (N) ER using dextran-coated charcoal (DCC) and filtration methods, respectively. To better define the intratissular distribution and content of ER, we also measured the presence of a 27 kDa heat shock protein (HSP27), a well established ER-associated protein, using D5 monoclonal antibody. Finally, we analysed the different molecular isoforms of both S and N ER using size exclusion-high performance liquid chromatography (SE-HPLC). High affinity (type I) sites of estrogen binding were detected in 17 out of 19 samples in either S or N fraction, although only 9 out of 19 cases displayed site 1 ER in both cell compartments. ER levels in aortic tissues, detected by radioligand method, compare well with those we have found in other hormone-sensitive human cancer tissues and cells. SE-HPLC analysis revealed two main receptor isoforms in the soluble fraction, having 65 kDa and 18 kDa molecular mass, while a minor component of 29 kDa was also found; the nuclear fraction displayed again two major components of 38 and 23 kDa. Using the HSP27 immunohistochemistry we observed a major staining occurring in smooth muscle cells (SMC), with an increasing intensity towards the lumen. All samples, including the ER negative ones, exhibited some degree of histochemical staining. Using an arbitrary cut-off value, 7 out of 12 samples displayed a highly positive staining, 6 of which showed nuclear ER. Furthermore, SE-HPLC separation indicated the presence of a 64.9 kDa component in the soluble fraction, according to the well known relative molecular mass of ER. Following HSP27 immunohistochemistry, the overall staining intensity in aortic SMC approaches that seen in endometrial and breast epithelia, whilst the muscle ER content is generally lower. Although our data are compatible with a direct role of estrogens in arterial function, the extent of the link with arterial disease remains to be established.     

Dose-dependent selective cytotoxicity of extracts from marine green alga, Cladophoropsis vaucheriaeformis, against mouse leukemia L1210 cells

The cases of four children who presented with respiratory distress unresponsive to bronchodilator treatment and who were subsequently diagnosed with severe anemia were retrospectively reviewed from charts at a children's hospital with 110 beds and an emergency department census of 32,000 per year. The four children, age range 11 months to 2 years, each initially presented with tachypnea. Three had wheezing, three were pale, and each received outpatient bronchodilator treatment. Three to 4 days later, each patient was hospitalized for continued respiratory distress. Each child was pale, and hemoglobin levels ranged from 2.5 to 5.2 g/dL. In three children, respiratory distress resolved with blood transfusions. Final diagnoses were transient erythroblastopenia of childhood in 2, acute lymphoblastic leukemia in 1, and iron deficiency anemia in 1. Severe anemia is a known cause of respiratory distress. These cases remind the clinician to consider severe anemia in children who are tachypneic, pale, and unresponsive to treatment of respiratory disorders.     

Reassignment of the gene encoding the Escherichia coli hydrogenase 2 small subunit--identification of a soluble precursor of the small subunit in a hypB mutant

An active tryptic fragment of hydrogenase 2 from Escherichia coli has been isolated from the periplasmic face of the cytoplasmic membrane, and the large and small subunits N-terminally sequenced. The large subunit is encoded by the hybC gene and shows no N-terminal processing, other than removal of the initiator methionine during its biosynthesis. Both N-terminal and the subsequent internal tryptic-fragment amino acid sequence indicate that the small subunit is neither encoded by hybA, a gene previously identified as encoding the small subunit [Menon et al. (1994) J. Bacteriol. 176, 4416-4423], nor any of the remaining genes in the hyb operon. Genome sequence analysis revealed the presence of an open reading frame which could potentially encode the peptide sequences of the proteolysed small subunit. The gene, designated hyb0, lies directly upstream of, and is separated by two nucleotides from, the start of the hybA gene. Hyb0, which shares an approximate 40% identity with other hydrogenase small subunit amino acid sequences, is synthesised with an N-terminal signal sequence containing a twin-arginine motif which is probably required for export of the enzyme. In the mature enzyme the small subunit is proteolytically cleaved after Ala37. Immunological analysis of strains overproducing either recombinant Hyb0 or HybA using antibodies specific for hydrogenase 2, readily identified Hyb0 as the small subunit. In a pleiotropic hypB mutant, which is unable to insert nickel into the active site, both the large and small subunits accumulate as unprocessed, soluble forms, consistent with the two subunits being assembled and processed in a coordinated manner during biosynthesis.