The expression, purification and crystallization of the epsilon subunit of the F1 portion of the ATPase of Escherichia coli.

The epsilon subunit of the F0F1-ATPase from Escherichia coli has been expressed in E. coli as a fusion protein with glutathione S-transferase from the parasitic helminth Schistosoma japonicum. The epsilon subunit released by thrombin treatment of the purified fusion protein carried two amino acid changes, A1G and M2S, and was obtained in a yield of about five milligrams per litre of cultured cells. The two amino acid changes were shown not to affect function. The protein has been crystallized in a form suitable for X-ray diffraction structure analysis. The crystals are hexagonal, space group P6(1)22 (or P6(5)22), with a = b = 94.9 A, c = 57.1 A and gamma = 120 degrees. The diffraction from small crystals extends to at least 2.9 A resolution.     

Immunocytochemical Localization of Phosphoribulose Kinase in the Cyanelles of Cyanophora paradoxa and Glaucocystis nostochinearum

The distribution of phosphoribulose kinase (PRK) in the cyanelles of Cyanophora paradoxa Korschikoff and Glaucocystis nostochinearum Itzigsohn was studied by protein A-gold immunoelectron microscopy. In both endocyanomes, antiserum against PRK heavily labeled the thylakoid region of the cyanelles, whereas little or no label was present over the carboxysomes. Antiserum against ribulose 1,5-bisphosphate carboxylase/oxygenase by contrast heavily labeled the carboxysomes of each endocyanome. In vitro studies of PRK distribution in cell-free extracts of C. paradoxa showed that 93% of the enzyme was in the soluble fraction. Quantitative immunoelectron microscopy showed that more than 99% of the PRK in the cyanelle of C. paradoxa was localized in the thylakoid region. We conclude that the carboxysomes of cyanelles like the carboxysomes of autotrophic prokaryotes and the pyrenoids of green algal chloroplasts do not contain phosphoribulose kinase.     

Use of a rapid bioluminescence assay for detecting cyanobacterial microcystin toxicity

The recent rise in the awareness of the occurrence of toxic cyanobacterial blooms in aquatic environments, with associated human health problems and animal deaths, has increased the need for rapid, reliable and sensitive methods of determining cyanobacterial toxicity. A luminescent bacterial toxicity test was assessed as a complement to the established mouse bioassay. Seventeen samples including pure cyanobacterial microcystin-LR hepatotoxin, laboratory isolates and natural blooms of cyanobacteria were tested and toxicity data compared with mouse LD50 values. Microcystin-LR and all five microcystin-containing cyanobacterial samples, hepatotoxic by mouse test gave EC50 values of less than 0.46 mg/ml in bioluminescence-based Microtox assays. Of 11 samples non-toxic by mouse bioassay, only two gave an EC50 of less than 0.98 mg/ml by bioluminescence assay. It is suggested that the Microtox bioluminescence assay may prove useful in the preliminary screening of cyanobacterial blooms for microcystin-based toxicity.     

Intranasal activity of the growth hormone releasing peptide His-D-Trp-Ala-Trp-D-Phe-Lys-NH2 in conscious dogs

This series of experiments was conducted to evaluate the growth hormone (GH) releasing activity of intranasally administered His-D-Trp-Ala-Trp-D-Phe-Lys-NH2 (GHRP-6, SK&F 110679) in conscious dogs. Intranasal administration of GHRP-6 increased plasma growth hormone levels in the conscious dog in a dose-related manner. Doses of 0.25 and 0.5 mg/kg produced GH levels of 11.3 +/- 4.8 ng/ml and 28.6 +/- 8.0 ng/ml, respectively. Peak levels were observed 15 minutes after dosing and GH levels were elevated for up to 105 minutes after intranasal dosing. Intranasal administration of isotonic saline did not produce any change in basal (negligable) GH levels. When GHRP-6 was given by the intravenous route, a maximal dose of 0.5 mg/kg, produced a peak plasma GH concentration of 60.8 +/- 10.5 ng/ml. Saline had no effect on GH levels when given intravenously. Using the intravenous and intranasal GH response data (i.e., area under the time-response curves), the intranasal bioavailability of GHRP-6 was estimated to be 34.4 to 44.9%. The results of these studies suggest that significant activity and excellent bioavailability can be achieved when GHRP-6 is administered by the intranasal route to conscious dogs. Based on these results, the intranasal activity of GHRP-6 should be evaluated in man. The successful intranasal administration of this peptide in man should provide GH therapy with reduced patient discomfort and better patient compliance when compared to presently available parenterally administered remedies.     

Purification of ribulose 1,5-bisphosphate carboxylase/oxygenase and of carboxysomes from Thiobacillus thyasiris the putative symbiont of Thyasira flexuosa (Montagu)

The bacterial symbionts of many marine invertebrates contain ribulose 1,5-bisphosphate (RuBP) carboxylase but apparently no carboxysomes, polyhedral bodies containing RuBP carboxylase. In the few cases where polyhedral bodies have been observed they have not been characterised enzymatically. Polyhedral bodies, 50–90 nm in diameter, were observed in thin cell sections of Thiobacillus thyasiris the putative symbiont of Thyasira flexuosa and RuBP carboxylase activity was detected in both soluble and particulate fractions after centrifugation of cell-free extracts. RuBP carboxylase purified 90-fold from the soluble fraction was of high molecular weight and consisted of large and small subunits, with molecular weights of 53,110 and 11,100 respectively. Particulate RuBP carboxylase activity was associated with polyhedral bodies 50–100 nm in diameter, as revealed by density gradient centrifugation and electron microscopy. Therefore, the polyhedral bodies were inferred to be carboxysomes. Native electrophoresis of isolated carboxysomes demonstrated a major band which comigrated with the purified RuBP carboxylase and three minor bands of lower molecular weight. Sodium dodecyl-sulphate (SDS) gel electrophoresis of SDS-dissociated carboxysomes demonstrated nine major polypeptides two of which were the large and small subunits of RuBP carboxylase. The RuBP carboxylase subunits represented 21% of the total carboxysomal protein. The most abundant polypeptide had a molecular weight of 40,500. Knowledge of carboxysome composition is necessary to provide an understanding of carboxysome function.Abbreviations FPLC fast performance liquid chromatography - IB isolation buffer - PAGE polyacrylamide gel electrophoresis - RuBP carboxylase - ribulose 1,5-bisphosphate carboxylase/oxygenase - SDS sodium dodecyl-sulphate     

Construction of a gene bank and identification of the ribulose bisphosphate carboxylase/oxygenase genes from the nutritionally versatile cyanobacterium Chlorogloeopsis fritschii

A gene bank of the nutritionally versatile, nitrogen-fixing cyanobacterium Chlorogloeopsis fritschii was constructed in Charon 4A. 2,800 recombinants containing 10–20 kbp C. fritschii DNA fragments were screened by Southern hybridization using probes containing the genes for the large (LSU) and small (SSU) subunits of ribulose bisphosphate carboxylase/oxygenase (RuBisCO) from Anacystis nidulans. A single recombinant plaque (CDG1) containing a 10.9 kbp EcoR1 fragment from C. fritschii hybridized to both the LSU and SSU probes, indicating a possible linkage of these RuBisCO genes in C. fritschii. RuBisCO activity and protein were detected in CDG1 lysates of Escherichia coli. Hybridization was also obtained between C. fritschii DNA and the LSU probe from Chlamydomonas reinhardtii, although no homology was detected using the LSU probe from maize or the SSU probe from pea.Abbreviations RuBisCO d-ribulose 1,5-bisphosphate carboxylase/oxygenase - RuBP d-ribulose 1,5-bisphosphate - LSU large subunit of RuBisCO - SSU small subunit of RuBisCO - SDS sodium dodecyl sulphate - DOC deoxycholate     

Phosphopyruvate carboxylase activity and carbon dioxide fixation via C4 acids over the division cycle in synchronized Euglena cultures

Summary Phosphoryruvate carboxylase activity was determined in division synchronized Euglena gracilis strain Z cultures. The profile of enzyme activity was essentially that of a peak enzyme; activity increased over the light phase of the cycle, doubling by early dark phase followed by a substantial decline in activity near the end of the dark phase. Dark carbon dioxide fixation did not parallel changes in phosphoryruvate carboxylase activity. The rate of carbon dioxide fixation increased fourfold over the light phase but decreased in the dark phase until it was only double the rate at the beginning of the light phase.Although the specific activity of phosphopyruvate carboxylase was greater than that of ribulose 1–5 diphosphate carboxylase in Euglena cell extracts at all stages over the division cycle C₄ acids were not an early product of carbon dioxide fixation in the light, neither did they ever account for more than a small proportion of the total ¹⁴C present in the soluble fraction of the cells. Phosphopyruvate carboxylase was shown by the non-aqueous localization technique to be present in the cytoplasm in Euglena, and it is concluded that the main function of this enzyme in algal cells is to provide an anaplerotic sequence to the tricarboxylic acid cycle.     

Accumulation of technetium by cyanobacteria

Accumulation of pertechnetate ions (⁹⁹TcO₄ ^–) by the cyanobacterial speciesSynechocystis PCC 6803,Synechococcus PCC 6301,Plectonema boryanum,Anabaena variabilis and a redOscillatoria sp. consisted solely of a single rapid energy-independent phase (biosorption); no energy-dependent uptake was detected. Biosorption of TcO₄ ^– was concentration-dependent and could be described by a Freundlich adsorption isotherm for each cyanobacterial species examined. Decreasing pH increased the accumulation of TcO₄ ^– by all the species as did an increase in external NaCl concentration. Accumulation of TcO₄ ^– was also increased inA. variabilis, P. boryanum and the redOscillatoria by an increased external osmotic potential. Concentrations of cations affected TcO₄ ^– accumulation; K⁺ increased accumulation in all the species, Mg²⁺, Ca²⁺, Sr²⁺ and Cs⁺ increased accumulation inSynechococcus PCC 6301 and Ca²⁺ increased accumulation by the redOscillatoria. Some anions decreased TcO₄ ^– accumulation; CO₃ ^2– inA. variabilis and the redOscillatoria, SO₄ ^2– inSynechocystis PCC 6803, and HCO₃ ^– inP. boryanum. The majority of TcO₄ ^– accumulated by all the cyanobacteria was easily desorbed, with no difference in the amounts desorbed between desorption agents of different pH or cation concentration.(*author for correspondence)     

A rapid whole-cell assay for superoxide dismutase

A procedure is described for the intact-cell assay of superoxide dismutase(s). The technique involves the use of toluene which renders the cells permeable to the necessary components of a photochemical assay for superoxide dismutase. Whole-cell superoxide dismutase activities from a number of procaryotic and eucaryotic microorganisms compare with cell-free activities and with activities reported in the literature. Using this procedure, changing levels of superoxide dismutase are readily monitored under conditions known to modulate superoxide dismutase activity assayed in vitro. In whole cells of Escherichia coli, exogenous methyl viologen causes a marked increase in superoxide dismutase activity, whereas in the cyanobacterium, Microcystis aeruginosa, such treatment leads to a marked, light-dependent loss of whole-cell superoxide dismutase activity.