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PubMed2002

Chlorella virus RNA triphosphatase. Mutational analysis and mechanism of inhibition by tripolyphosphate.

Chlorella virus RNA triphosphatase (cvRtp1) is the smallest member of a family of metal-dependent phosphohydrolases that includes the RNA triphosphatases of fungi, protozoa, poxviruses, and baculoviruses. The primary structure of cvRtp1 is more similar to that of the yeast RNA triphosphatase Cet1 than it is to the RNA triphosphatases of other DNA viruses. To evaluate the higher order structural similarities between cvRtp1 and the fungal enzymes, we performed an alanine scan of individual residues of cvRtp1 that were predicted, on the basis of the crystal structure of Cet1, to be located at or near the active site. Twelve residues (Glu(24), Glu(26), Asp(64), Arg(76), Lys(90), Glu(112), Arg(127), Lys(129), Arg(131), Asp(142), Glu(163), and Glu(165)) were deemed essential for catalysis by cvRtp1, insofar as their replacement by alanine reduced phosphohydrolase activity to <5% of the wild-type value. Structure-activity relationships were elucidated by introducing conservative substitutions at the essential positions. The mutational results suggest that the active site of cvRtp1 is likely to adopt a tunnel fold like that of Cet1 and that a similar constellation of side chains within the tunnel is responsible for metal binding and reaction chemistry. Nonetheless, there are several discordant mutational effects in cvRtp1 versus Cet1, which suggest that different members of the phosphohydrolase family vary in their reliance on certain residues within the active site tunnel. We found that tripolyphosphate and pyrophosphate were potent competitive inhibitors of cvRtp1 (K(i) = 0.6 microm tripolyphosphate and 2.4 microm pyrophosphate, respectively), whereas phosphate had little effect. cvRtp1 displayed a weak intrinsic tripolyphosphatase activity (3% of its ATPase activity) but was unable to hydrolyze pyrophosphate.

Acid Anhydride Hydrolases↗
PubMed1974

The hexose-proton cotransport system of chlorella. pH-dependent change in Km values and translocation constants of the uptake system.

The proton concentration in the medium affects the maximal velocity of sugar uptake with a K(m) of 0.3 mM (high affinity uptake). By decreasing the proton concentration a decrease in high affinity sugar uptake is observed, in parallel the activity of a low affinity uptake system (K(m) of 50 mM) rises. Both systems add up to 100%. The existence of the carrier in two conformational states (protonated and unprotonated) has been proposed therefore, the protonated form with high affinity to 6-deoxyglucose, the unprotonated form with low affinity. A plot of extrapolated V(max) values at low substrate concentration versus proton concentration results in a K(m) for protons of 0.14 microM, i.e. half-maximal protonation of the carrier is achieved at pH 6.85. The stoichiometry of protons cotransported per 6-deoxyglucose is close to 1 at pH 6.0-6.5. At higher pH values the stoichiometry continuously decreases; at pH 8.0 only one proton is cotransported per four molecules of sugar. Whereas the translocation of the protonated carrier is strictly dependent on sugar this coupling is less strict for the unprotonated form. Therefore at alkaline pH a considerable net efflux of accumulated sugar can occur. The dependence of sugar accumulation on pH has been measured. The decrease in accumulation with higher pH values can quantitatively be explained by the decrease in the amount of protonated carrier. The properties of the unprotonated carrier resemble strikingly the properties of carrier at the inner side of the membrane. The inside pH of Chlorella was measured with the weak acid 5,5-dimethyl-2, 4-oxazolidinedion (DMO). At an outside pH of 6.5 the internal pH was found to be 7.2. To explain the extent of sugar accumulation it has to be assumed that the membrane potential also contributes to active sugar transport in this alga.

Biological Transport↗
PubMed1998

Chlorella virus DNA ligase: nick recognition and mutational analysis.

Chlorella virus PBCV-1 DNA ligase seals nicked DNA substrates consisting of a 5'-phosphate-terminated strand and a 3'-hydroxyl-terminated strand annealed to a bridging DNA template strand. The enzyme discriminates at the DNA binding step between substrates containing a 5'-phosphate versus a 5'-hydroxyl at the nick. Mutational analysis of the active site motif KxDGxR (residues 27-32) illuminates essential roles for the conserved Lys, Asp and Arg moieties at different steps of the ligase reaction. Mutant K27A is unable to form the covalent ligase-(Lys-straightepsilonN-P)-adenylate intermediate and hence cannot activate a nicked DNA substrate via formation of the DNA-adenylate intermediate. Nonetheless, K27A catalyzes phosphodiester bond formation at a pre-adenylated nick. This shows that the active site lysine is not required for the strand closure reaction. K27A binds to nicked DNA-adenylate, but not to a standard DNA nick. This suggests that occupancy of the AMP binding pocket of DNA ligase is important for nick recognition. Mutant D29A is active in enzyme-adenylate formation and binds readily to nicked DNA, but is inert in DNA-adenylate formation. R32A is unable to catalyze any of the three reactions of the ligation pathway and does not bind to nicked DNA.

Adenosine Monophosphate↗
PubMed2002

Role of nucleotidyltransferase motifs I, III and IV in the catalysis of phosphodiester bond formation by Chlorella virus DNA ligase.

ATP-dependent DNA ligases catalyze the sealing of 5'-phosphate and 3'-hydroxyl termini at DNA nicks by means of a series of three nucleotidyl transfer steps. Here we have analyzed by site-directed mutagenesis the roles of conserved amino acids of Chlorella virus DNA ligase during the third step of the ligation pathway, which entails reaction of the 3'-OH of the nick with the DNA-adenylate intermediate to form a phosphodiester and release AMP. We found that Asp65 and Glu67 in nucleotidyltransferase motif III and Glu161 in motif IV enhance the rate of step 3 phosphodiester formation by factors of 20, 1000 and 60, respectively. Asp29 and Arg32 in nucleotidyltransferase motif I enhance the rate of step 3 by 60-fold. Gel shift analysis showed that mutations of Arg32 and Asp65 suppressed ligase binding to a pre-adenylated nick, whereas Asp29, Glu67 and Glu161 mutants bound stably to DNA-adenylate. We infer that Asp29, Glu67 and Glu161 are involved directly in the step 3 reaction. In several cases, the effects of alanine or conservative mutations on step 3 were modest compared to their effects on the composite ligation reaction and individual upstream steps. These results, in concert with available crystallographic data, suggest that the active site of DNA ligase is remodeled during the three steps of the pathway and that some of the catalytic side chains play distinct roles at different stages.

Adenosine Monophosphate↗
PubMed1993

Heme Inhibition of [delta]-Aminolevulinic Acid Synthesis Is Enhanced by Glutathione in Cell-Free Extracts of Chlorella.

In plants, algae, and many bacteria, the heme and chlorophyll precursor, [delta]-aminolevulinic acid (ALA), is synthesized from glutamate in a reaction involving a glutamyl-tRNA intermediate and requiring ATP and NADPH as cofactors. In particulate-free extracts of algae and chloroplasts, ALA synthesis is inhibited by heme. Inclusion of 1.0 mM glutathione (GSH) in an enzyme and tRNA extract, derived from the green alga Chlorella vulgaris, lowered the concentration of heme required for 50% inhibition approximately 10-fold. The effect of GSH could not be duplicated with other reduced sulfhydryl compounds, including mercaptoethanol, dithiothreitol, and cysteine, or with imidazole or bovine serum albumin, which bind to heme and dissociate heme dimers. Absorption spectroscopy indicated that heme was fully reduced in incubation medium containing dithiothreitol, and addition of GSH did not alter the heme reduction state. Oxidized GSH was as effective in enhancing heme inhibition as the reduced form. Co-protoporphyrin IX inhibited ALA synthesis nearly as effectively as heme, and 1.0 mM GSH lowered the concentration required for 50% inhibition approximately 10-fold. Because GSH did not influence the reduction state of heme in the incubation medium, and because GSH could not be replaced by other reduced sulfhydryl compounds or ascorbate, the effect of GSH cannot be explained by action as a sulfhydryl protectant or heme reductant. Preincubation of enzyme extract with GSH, followed by rapid gel filtration, could not substitute for inclusion of GSH with heme during the reaction. The results suggest that GSH must specifically interact with the enzyme extract in the presence of the inhibitor to enhance the inhibition.

Journal Article↗
PubMed1993

Identification of Distinct Internal and External Isozymes of Carbonic Anhydrase in Chlorella saccharophila.

External carbonic anhydrase (CA) was detected in whole cells of alkaline-grown Chlorella saccharophila but was suppressed by growth at acid pH or growth on elevated levels of CO2. Internal CA activity was measured potentiometrically as an increase in activity in cell extracts over that of intact cells. Cells grown under all conditions had equal levels of internal CA activity. Two isozymes were identified after electrophoretic separation of soluble proteins on cellulose acetate plates. The fast isozyme was found in cells grown under all conditions, whereas the slow isozyme was found only in cells grown at alkaline pH. Western blot analysis following sodium dodecyl sulfate-polyacrylamide gel electrophoresis using antibodies produced against the periplasmic form of CA from Chlamydomonas reinhardtii revealed a single band at 39 kD, which did not change in intensity between growth conditions and was associated only with proteins eluted from the fast band. The slow isozyme was inactivated by incubation of cell extract at 30[deg]C and by incubation in 10 mM dithiothreitol, whereas the internal form was unaffected. These results indicate that external and internal forms of CA differ in structure and their activities respond differently to environmental conditions.

Journal Article↗
PubMed1995

Quantification of the Contribution of CO2, HCO3-, and External Carbonic Anhydrase to Photosynthesis at Low Dissolved Inorganic Carbon in Chlorella saccharophila.

An equation has been developed incorporating whole-cell rate constants for CO2 and HCO3- that describes accurately photosynthesis (Phs) in suspensions of unicellular algae at low dissolved inorganic carbon. At pH 8.0 the concentration of CO2 available to the algal cells depends on the rate of supply from, and the loss to, HCO3- and the rate of use by the cells. At elevated cell densities (>30 mg chlorophyll [Chl] L-1), at which CO2 use by the cells is high, the slope of a graph of absolute Phs versus Chl concentration approaches the rate of Phs on a milligram of Chl basis because of HCO3- use alone. The slope of a graph of Phs versus HCO3- will be the rate constant for HCO3-, and for Chlorella saccharophila it was 0.16 L mg-1 Chl h-1. The difference between the constants for dissolved inorganic carbon (measured in cells with external carbonic anhydrase) and HCO3-1 is the constant for CO2, which was 26 L mg-1 Chl h-1. This difference causes the half-saturation constant for Phs to increase 5- to 6-fold at high cell densities. The increase in CO2 use as a result of external carbonic anhydrase is described mathematically as a function of cell density.

Journal Article↗
PubMed1995

Induction of CO2 and Bicarbonate Transport in the Green Alga Chlorella ellipsoidea (I. Time Course of Induction of the Two Systems).

Changes in the physiological properties of the green alga Chlorella ellipsoidea (UTEX 20) were determined during adaptation from high CO2 to air. Cells of C. ellipsoidea, grown in high CO2, had an extremely low affinity for dissolved inorganic carbon (DIC). However, high-affinity DIC transport was induced rapidly after switching to air, which caused a massive decrease in the DIC concentration in the medium. Rates of O2 evolution without added carbonic anhydrase (CA) were compared with calculated rates of uncatalyzed CO2 formation in the medium as a measure of active HCO3-uptake. Cells were found to be able to use HCO3- after 5 h of adaptation and this capacity increased during the next 17 h. The stimulation of O2 evolution upon CA addition was used as a measurement of active CO2 transport: such stimulation occurred 2 h after transfer and increased during the next 5 h. Increases in O2 evolution rates were correlated closely with an increasing capacity to accumulate intracellular pools of acid-labile DIC and with decreases in K1/2(CO2) and CO2-compensation point of the cells. Treatment of cells with cycloheximide (5 [mu]g mL-1) during adaptation completely inhibited DIC transport induction, whereas treatment with chloramphenicol (400 [mu]g mL-1) had no effect, indicating the requirement for cytoplasmic protein synthesis in the induction. These results suggest that both CO2 and HCO3- transport are induced upon transfer of cells from high CO2 to air and that there is a temporal separation between the induction of the two systems.

Journal Article↗
PubMed1995

Induction of CO2 and Bicarbonate Transport in the Green Alga Chlorella ellipsoidea (II. Evidence for Induction in Response to External CO2 Concentration).

The critical species and concentrations of dissolved inorganic carbon (DIC) required for the induction of DIC transport during adaptation to low CO2 were determined for the green alga Chlorella ellipsoidea. The concentration of dissolved CO2 needed for the induction of both CO2 and HCO3- transport was independent of pH during adaptation, whereas the total DIC concentration required increased at alkaline pH. At pH 7.5, the minimum equilibrium DIC concentration at which high CO2 characteristics were maintained, i.e. transport was repressed, was 2100 [mu]M, whereas the maximum equilibrium DIC concentration below which DIC transport was fully induced (DICIND) was 500 [mu]M. Intracellular DIC concentration during adaptation to DICIND decreased temporarily after 2 h to 60% of the maximum level but recovered after 3 h of adaptation. After 3 h of adaptation to DICIND, cells exhibited maximum O2 evolution rate at DICIND. When cells partially adapted to DICIND were returned to high CO2, there was an immediate halt to the induction of transport and a gradual decrease in transport capacity over 23 h. The capacity for the induction of transport was unaffected by the absence of light. These results indicate that changes in the internal DIC pool during adaptation to low CO2 do not trigger the induction of DIC transport and that the induction is not light dependent. Induction of DIC transport in C. ellipsoidea appears to occur in response to the continuous exposure of cells to a critical CO2 concentration in the external medium.

Journal Article↗
PubMed1996

Photosystem II Excitation Pressure and Photosynthetic Carbon Metabolism in Chlorella vulgaris.

Chlorella vulgaris grown at 5[deg]C/150 [mu]mol m-2 s-1 mimics cells grown under high irradiance (27[deg]C/2200 [mu]mol m-2 s-1). This has been rationalized through the suggestion that both populations of cells were exposed to comparable photosystem II (PSII) excitation pressures measured as the chlorophyll a fluorescence quenching parameter, 1 - qP (D.P. Maxwell, S. Falk, N.P.A. Huner [1995] Plant Physiol 107: 687-694). To assess the possible role(s) of feed-back mechanisms on PSII excitation pressure, stromal and cytosolic carbon metabolism were examined. Sucrose phosphate synthase and fructose-1,6-bisphosphatase activities as well as the ratios of fructose-1,6-bisphosphate/fructose-6-phosphate and sucrose/starch indicated that cells grown at 27[deg]C/2200 [mu]mol m-2 s-1 appeared to exhibit a restriction in starch metabolism. In contrast, cells grown at 5[deg]C/150 [mu]mol m-2 s-1 appeared to exhibit a restriction in the sucrose metabolism based on decreased cytosolic fructose-1,6- bisphosphatase and sucrose phosphate synthase activities as well as a low sucrose/starch ratio. These metabolic restrictions may feed-back on photosynthetic electron transport and, thus, contribute to the observed PSII excitation pressure. We conclude that, although PSII excitation pressure may reflect redox regulation of photosynthetic acclimation to light and temperature in C. vulgaris, it cannot be considered the primary redox signal. Alternative metabolic sensing/signaling mechanisms are discussed.

Journal Article↗
PubMed1966

Responses of Heterotrophic Cultures of Chlorella vulgaris Beyerinck to Darkness and Light. II. Action Spectrum for and Mechanism of the Light Requirement for Heterotrophic Growth.

Chlorella vulgaris Beyerinck (Emerson's strain), fails to grow in the dark even when sugars are provided. This phenomenon was clearly demonstrated in the alga, C. vulgaris, for which the growth rate in darkness on a glucose medium remained constant for 2 days and then declined to approach zero. Pigment concentrations also declined in darkness. Changes in flow rate of 1% CO(2)-in-air from zero to 7 ml per minute caused a progressive increase in the dark growth rate over a 5-day period, but did not maintain growth in the dark. Rates above 7 ml per minute produced no changes in growth rates.White light intensities below the compensation point of the alga maintained heterotrophic growth. The saturation value for this response was 0.8 muw/cm(2). White light also initiated growth in nongrowing cultures transferred from darkness to light.The action spectrum for heterotrophic growth indicated a porphyrin as the active pigment. Light in the 425 mmu region was 4 times as effective as white light in stimulating heterotrophic growth. A secondary peak of growth stimulation occurred in the 575 mmu region.The respiration of glucose by the alga was stimulated by low intensities of white light. This response was not immediate, but was clearly present after the third day of incubation.Malonate and cyanide were inhibitory to growth of C. vulgaris on inorganic medium or glucose medium under 300 ft-c of white light. These data suggested that succinic dehydrogenase and cytochrome oxidase systems were present.Substances inhibitory to growth were excreted into the medium under dark-growth conditions, and 2 of these substances were indentified as formic and acetic acids.The evidence suggested that respiration of glucose cannot proceed for an extended period of time in darkness. The reason for this is postulated to be the lack of a cytochrome or a cytochrome precursor.

Journal Article↗
PubMed1966

Monochromatic light saturation curves for photosynthesis in Chlorella.

We used a small oxygen electrode and a grating monochromator of 10 mmu half-band width to determine light-saturation curves of photosynthesis for films of Chlorella pyrenoidosa no more than 1 cell thick. All curves were referenced to the lightsaturated rate observed in 5 mw/cm(2) of 680 mmu. To a close approximation (+/- 2%) the light-saturated rate was independent of wavelength over the region in which light intensity was sufficient to make the test (450-705 mmu). At wavelengths of high absorption we obtained intensities sufficient to cause photoinhibition. As a measure of photoinhibition we used the breakpoint, the lowest intensity at which rate of photosynthesis decreased with time. In general, the saturated rate was slightly lower and the estimated breakpoint was considerably lower at wavelengths of high absorption. Maximum rate of absorption of quanta at the breakpoint was highest in the far-red (>/= 700 mmu), lower and relatively constant in the near-red (630-680 mmu), and lowest in the blue. At 435 and 450 mmu the breakpoint occurred below saturation. We attribute small deviations in maximum rate of photosynthesis to effects of photoinhibition. Independence of wavelength observed for the light-saturated rate is consistent with models of photosynthesis in which maximum rate is limited by the same dark reaction at all wavelengths.

Eukaryota↗
PubMed1966

Chlorophyll synthesis in chlorella I. Occurrence of a lag phase on initiation of a dilute culture.

A culture of Chlorella established by 30-fold dilution of a culture already grown to a level of 15 ml packed cell volume per liter produces little chlorophyll for approximately 12 hours. Investigation of other characteristics such as nitrogen incorporation, increase in packed cell volume and dry weight as well as RNA level show all of these to increase without any significant lag. alpha-Linolenate, which can be considered as a chloroplast marker, increased markedly. Photosynthetic oxygen evolution and respiration as well as the heme enzyme, catalase, increase also, indicating that the lag in chlorophyll synthesis is not due to a general inability to produce the porphyrin moiety.The lag period was demonstrated not to be related to a deficiency of culture elements nor to inhibitors produced. Neither was there a need for a conditioning period of the medium.It is concluded that the lag in chlorophyll synthesis is unique and that the reasons for it are related to the response of the cells to environmental conditions following dilution.

Journal Article↗
PubMed1966

Chlorophyll Synthesis in Chlorella II. Effect of Glucose and Light Intensity on the Lag Phase.

Dense cultures of Chlorella grown under our conditions contain large amounts of chlorophyll on a packed cell basis. Upon dilution the cells are, in effect, put into an environment in which light is not limiting and chlorophyll seemingly in excess (at least temporarily). Thus it is not economical for the cell to produce more chlorophyll until it is the limiting factor. It is our contention that the cell may sense this situation by the piling up of products such as glucose or starch. The observed light intensity effect which causes a stimulation of chlorophyll production at lowered intensities is analogous to that previously reported by others and is felt to be related to the rate of photosynthate production.Chlorophyll synthesis commences after a period of about 12 hours. Once it has become established, added substrate or light have a smaller effect. At 36 hours addition of glucose stimulates growth markedly but chlorophyll production continues at the control rate. This suggests that the effect of substrate is not upon the action of an enzyme involved in the actual synthesis of the chlorophyll molecule. Glucose has been known to influence production of certain enzymes involved usually in substrate catabolism. It is quite possible that the role of glucose here is quite similar. The data are consistent with such an explanation. The substrate would have to influence the production of an enzyme in the magnesium branch of the biosynthetic pathway of porphyrin compounds and not in the portion common to both iron and magnesium porphyrins. Accordingly the lag period is viewed as both an inhibitory period as well as a preparatory period for subsequent chlorophyll synthesis.

Journal Article↗
PubMed1966

Methionine transport in Chlorella vulgaris.

Absorption of (35)S-l-methionine by Chlorella vulgaris was measured at concentrations that ranged from 0.1 to 10.0 mumoles/ml. A brief, rapid phase of uptake was followed by a more prolonged, slower phase that was linear only at the lowest concentrations. The radioactivity accumulated by the end of 1 hour's incubation at an exogenous level of 0.1 mumole/ml was retained by the cells despite the inclusion of 10 mumoles/ml of nonradioactive methionine in the rinse medium. As the exogenous concentration was raised, the ratio of intracellular soluble radioactivity to exogenous radioactivity decreased. Analysis of the accumulated, soluble radioactivity showed that 90% was in the form of methionine and that about 10% had been converted to a compound with properties of S-adenosylmethionine. Azide and ethionine were the most effective of the inhibitors tested.

Biological Transport, Active↗
PubMed1966

Laser activation of rapid absorption changes in spinach chloroplasts and chlorella.

The kinetics of the 520 mmu absorption change in spinach chloroplasts and Chlorella vulgaris following a flash from the ruby laser have been determined as follows: rise halftime </= 0.3 x 10(-6) second; rapid recovery halftime = 5 to 6 x 10(-6) second; intermediate recovery halftime = 4 x 10(-4) second (spinach chloroplasts only); slow recovery halftime = 12 to 170 x 10(-3) second, dependent on the measuring light intensity and aerobicity of the suspension.The rapid phase of the 520 mmu reaction is approximately independent of temperature, from 295 degrees to 77 degrees Absolute.With increasing oxygenation of the sample, the extent of the rapid phase decreases, the extent of the slow phase increases, while the extent of the intermediate phase in spinach chloroplasts remains constant.In spinach chloroplasts, no recovery halftime of the 3 recovery phases for the 520 mmu absorption change was observed to correspond to the halftime for oxidation of cytochrome f (t((1/2)) = 1.3 x 10(-3) second).

Journal Article↗
PubMed1967

Alpha-linoleate and photosynthetic activity in Chlorella protothecoides.

Green cells of Chlorella protothecoides grown in nitrogen-rich low glucose media may be reversibly transformed to entirely chlorophyll-less cells in low nitrogen high glucose media. Photosynthetic rates and fatty ester compositions were determined during light and dark bleaching and during greening. Linolenic acid content remained unchanged during greening or bleaching. During light greening chlorophyll content and photosynthetic activity increased while oleic acid content decreased dramatically. As a result, the percent composition of linolenate appeared to parallel photosynthetic capability. Implication of alpha-linolenate in oxygen production, therefore, can not be based upon fatty acid percentage composition data alone.

Eukaryota↗
PubMed1967

Action spectrum for an enhancement of endogenous respiration by light in chlorella.

The oxygen consumption of a starved chlorophyll-free, yellow mutant of Chlorella vulgaris is enhanced by very small amounts of blue light (lambda 450 mmu); a saturation level is reached at about 500 ergs cm(-2) sec(-1). At that intensity the respiration is about 3 times greater than in the dark. An action spectrum for the enhancement of respiration shows 2 peaks around lambda 450 and 375 mmu. Flavins and cis-carotenoids are discussed as the pigments involved.

Journal Article↗