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PubMed1973

Partial Purification of the NADH-Nitrate Reductase Complex from Chlorella pyrenoidosa.

The nitrate reductase complex from Chlorella pyrenoidosa has been purified by a procedure which includes as main steps, ammonium sulfate fractionation, polyethylene glycol treatment, and DEAE-cellulose chromatography. The Michaelis constants for NADH, FAD, and NO(3) (-) in the NADH-nitrate reductase assay are 10 mum, 2.6 mum, and 0.23 mm, respectively. Heat treatment exerts varying effects on the enzymatic activities associated with the nitrate reductase complex.

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PubMed1973

Triparanol Inhibition of Sterol Biosynthesis in Chlorella ellipsoidea.

The sterol composition of C. ellipsoidea was markedly changed when this alga was grown in the presence of 1 mug/g triparanol. Triparanol appears to inhibit the removal of 14alpha-methyl group, the second alkylation at C-24, Delta(7)-reductase, and Delta(8) --> Delta(7)-isomerase. The effect of triparanol in Chlorella is much more diversified than the specific effect originally assigned to it in animals.

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PubMed1973

Sequence of Formation of Phosphoglycolate and Glycolate in Photosynthesizing Chlorella pyrenoidosa.

In Chlorella pyrenoidosa which have been photosynthesizing in either 1.5% (14)CO(2) or 0.05% (14)CO(2) in air, gassing with 100% O(2) results in rapid formation of phosphoglycolate which is apparently converted to glycolate. However, only about one-third to one-half of the rate of glycolate formation can be accounted for by this route. The remaining glycolate formation may be the result of the oxidation of sugar monophosphates. The rates of formation of both glycolate and phosphoglycolate are about four times greater with algae that have been photosynthesizing in 1.5% (14)CO(2) than with algae which have been photosynthesizing with air, when the algae are then gassed with 100% O(2).

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PubMed1973

Proline Uptake and Utilization by Chlorella pyrenoidosa.

Conditions for proline uptake and utilization by Chlorella pyrenoidosa Chick are described. Proline is taken up by growing cultures during late log phase growth after depletion of glucose from the medium. However, proline uptake by stationary phase cultures requires the presence of glucose in the medium. The results are consistent with the interpretation that some carbohydrate is required for proline uptake but proline uptake is inhibited by the accumulation of intracellular carbohydrates.Metabolism of proline was by incorporation into protein and oxidation to CO(2) by conversion to glutamic acid and subsequent metabolism of the carbon by the Krebs cycle.

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PubMed1974

Regulation of Hexose Transport in Chlorella vulgaris: Characteristics of Induction and Turnover.

Of nine species of unicellular algae tested, Chlorella vulgaris showed the highest inducibility for an active hexose transport system. Whereas the rate of uptake in all other species was increased by induction less than 5-fold, it was increased more than 400-fold in one strain of C. vulgaris. With glucose as inducer, the minimum time necessary to synthesize inducible proteins of the transport system was 15 minutes. The Km for induction with glucose is 5 mum and with 6-deoxyglucose 1 mm. The inducing sugars have to penetrate the cells to be effective.Evidence indicating that regulation of induction occurs at the transcriptional level was obtained. The induction was inhibited by 6-methylpurine. When cells were exposed to induce in the presence of actidione no increase in transport activity could be measured. After removal of actidione as well as the inducer, an increased uptake activity was observed after 30 to 60 minutes. The induced uptake system showed a turnover with a half-life of 4 to 6 hours at 26 C under nongrowing conditions; at 0 C turnover was negligible. Turnover was partly inhibited by anaerobic condition and by actidione; it was accelerated under growing conditions.

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PubMed1974

Inhibition of Sterol Biosynthesis in Chlorella sorokiniana by Triparanol.

When Chlorella sorokiniana was cultured in the presence of 1 mg/1 triparanol succinate, there was a 42% reduction in total sterol concentration. Algal biomass was reduced by approximately the same amount. In addition to the cycloartenol, cyclolaudenol, 24-methyl-pollinastanol, ergosta-5, 7-dien-3beta-ol, and ergosterol that occur in control culture, pollinastanol, 14alpha-methyl-5alpha-ergost-8-en-3beta-ol, 5alpha-ergosta-8, 14, 22-trien-3beta-ol, 5alpha-ergosta-8(14), 22-dien-3beta-ol, 5alpha-ergosta-8(9), 22-dien-3beta-ol, 5alpha-ergosta-8, 14-dien-3beta-ol, 5alpha-ergost-8(9)-3n-3beta-ol, 5alpha-ergost-8(14)-en-3beta-ol, 5alpha-ergosta-7, 22-dien-3beta-ol, and 5alpha-ergost-7-en-3beta-ol were isolated and identified from triparanol succinate-treated cells. A biosynthetic pathway for sterol biosynthesis in this organism is postulated based on all the sterols that were isolated and identified in triparanol-treated cultures of C. sorokiniana. Cyclolaudenol appears to be the product of the first alkylation at C-24 in this organism rather than the more common 24-methylene cycloartanol. Since 24-methylene sterols are needed for the second alkylation reaction, this would explain the absence of C-29 sterols in C. sorokiniana. Four of the sterols identified in C. sorokiniana are reported for the first time in a living organism. They are: 24-methyl pollinastanol, 5alpha-ergosta-8, 14, 22-trien-3beta-ol, 5alpha-ergosta-8(14), 22-dien-3beta-ol and 5alpha-ergost-8(14)-en-3beta-ol.

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PubMed1974

Nicotinamide Adenine Dinucleotide-dependent Proline Dehydrogenase in Chlorella.

An NAD-linked dehydrogenase from Chlorella pyrenoidosa Chick catalyzing the conversion of l-proline to Delta(1)-pyrroline-5-carboxylic acid was partially purified. Delta(1)-Pyrroline-5-carboxylic acid was identified as the product by co-chromatography of it and its o-aminobenzaldehyde derivative with authentic compounds. The enzyme is NAD and l-proline specific and is not an oxidase; NADP is not inhibitory. The Michaelis constant for NAD is 0.08 mm and for proline is 0.73 mm.

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PubMed1974

Control of Free Methionine Production in Wild Type and Ethionine-resistant Mutants of Chlorella sorokiniana.

Mutants of Chlorella sorokiniana selected for resistance to the methionine analogue ethionine took up ethionine at the same rate as did the wild type strain. Cells of two ethionine-resistant mutants produced severalfold higher levels of free methionine and cysteine than did wild type cells.Exogenous methionine had no apparent effect on free methionine production in a mutant that produces excessive levels of free methionine. Under the same conditions, production of free methionine was relatively inhibited in wild type cells and in a mutant that produces wild type levels of free methionine.The results suggest that free methionine production in the wild type strain is subject to endproduct control, and that this control is lacking in one class of ethionine-resistant mutants.

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PubMed1974

The Respiratory Chain of Chlorella protothecoides: I. Inhibitor Responses and Cytochrome Components of Whole Cells.

The respiration and cytochrome properties of "glucose-bleached" Chlorella protothecoides Krüger, Indiana strain 25, were studied. This organism, when grown heterotrophically with high glucose and a low organic nitrogen source, has no chlorophyll, little carotenoid, and diminished chloroplast structure-factors which make it suitable for respiration studies.Whole cell endogenous oxygen uptake rates are either stimulated or only slightly inhibited by cyanide, azide, CO, and antimycin. When these inhibitors are used with m-chlorobenz-hydroxamic acid (mCLAM), an inhibitor of higher plant mitochondrial alternate oxidase, O(2) uptake is inhibited. There is little effect of mCLAM by itself on the rate of oxygen uptake. The inhibition by CO of O(2) uptake in the presence of mCLAM is reversed by light.The cytochrome chain of C. protothecoides consists of cytochromes aa(3), b, and c, as revealed by room temperature difference spectra. In common with mitochondria of higher plants, there is a further reduction of cytochrome b with dithionite. In the presence of antimycin, the cytochromes aa(3) and c are oxidized and cytochrome b is reduced. Cyanide causes a partial reduction of cytochromes aa(3) and c while cytochrome b remains oxidized. This general response is characteristic of higher plant mitochrondria having large amounts of cyanide-resistant respiration. Carbon monoxide spectra reveal one CO-combining pigment. The cytochrome b region differs from that of higher plants in that the typical complex spectrum does not appear at low temperature (-190 C).The concentration of cytochrome aa(3) per cell volume was observed during the greening of "glucose-bleached" cells. The concentration of these cytochromes nearly tripled during the 24 hours of the initial stages of greening.

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PubMed1974

Effects of cation levels of the nutrient medium on the biochemistry of chlorella: I. Concentration series.

The effects of variations in nutrient cation levels on the growth and biochemistry of Chlorella were investigated. This study involved concentration-series experiments in which the levels of Mg(2+), K(+), and Ca(2+) varied from deficiency to toxicity levels for growth. The nutrient sufficiency concentrations of Mg(2+) and K(+) were 0.08 and 0.10 meq/1, respectively. Deficiencies of Mg(2+) or K(+) reduced the growth rate, as well as cellular total nitrogen and unsaturated fatty acid levels. K(+) deficiency increased total lipid levels, while total fatty acids were unaffected. Increasing Mg(2+) or K(+) concentrations in the nutrient media were accompanied by corresponding increases in growth rate and certain biochemical fractions. Calcium was without effect except at a toxicity level. Cellular sufficiency concentrations for Mg(2+) and K(+) were 0.3 and 1.2% of the dry weight, respectively.

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PubMed1974

Effects of Cation Levels of the Nutrient Medium on the Biochemistry of Chlorella: II. Factorial Experiment.

A factorial experiment was designed to study the effects of Mg(2+), K(+), and Na(+) on the growth and biochemistry of Chlorella sorokiniana. Raising Mg(2+) or K(+) concentration in the nutrient medium increased growth rates as well as total N levels and Mg(2+) and K(+) accumulation by the cells. The total N effect was Mg(2+)-dependent-if Mg(2+) was below a certain level in the medium-increasing the K(+) concentration did not raise the total N level of cells. Low nutrient levels of K(+) decreased the levels of unsaturated fatty acids (especially 18:1 and 18:3), while increasing the levels of palmitic acid (16:0), total fatty acids, and total lipid. Increasing nutrient K(+) concentrations were accompanied by increases in levels of some unsaturated fatty acids, with a concomitant reduction in 16:0, total fatty acids and total lipid. Low Mg(2+) levels in the nutrient medium reduced the cellular levels of palmitic acid, total fatty acids, total lipid, and certain unsaturated fatty acids (though this last effect also depended on the nutrient level of K(+)). These relationships indicate that Mg(2+) may be important in the initial steps of fatty acid synthesis, whereas K(+) may be necessary for the formation of certain unsaturated fatty acids. Variations in Na(+) concentration did not have any significant effect on the growth and biochemistry of C. sorokiniana.

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PubMed1975

Ozone-induced Fatty Acid and Viability Changes in Chlorella.

Ozone-treated cells of the photosynthetic green alga Chlorella sorokiniana var. pacificensis exhibit an exponential decline in viability, as measured by their ability to form colonies on agar plates. Postexposure conditions appear to have little, if any, effect on this rate of decline. Except in young (early exponential phase) cells, culture age did not affect this rate. The decline in cell viability was correlated with the production of malondialdehyde, arising from the oxidative breakdown of an ozonide of unsaturated fatty acid material. The loss of fatty acids is substantiated by gas-liquid chromatography. A loss of 5 x 10(-15) moles of fatty acid per cell corresponds to 75% nonviable cells after 50 minutes of ozone exposure.

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PubMed1975

Growth of Chlorella in a Nitrate-limited Chemostat.

Chlorella pyrenoidosa was grown in a continuous-flow chemostat under nitrogen-limited conditions. The population density tended to oscillate very significantly. Net specific growth rate was only approximately a hyperbolic function of nitrate concentration in the chemostat. The best estimate of the half-saturation constant for nitrate is 6 mug of nitrogen per liter and it is unlikely that the value is greater than 14 mug per liter or 1 mum nitrate.The dry weight production of cells per unit of nitrogen taken up is a linearly decreasing function of the net specific growth rate with a maximum of 27.1 mg per mg N and a minimum of about 9 mg per mg N. Thus there is considerable storage of nitrogen at high growth rates. Both the dark respiration rate and the rate of photosynthesis at light saturation increase with increasing net specific growth rate.

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PubMed1975

Purification and Some Properties of Chlorella fusca Ribulose 1,5-Diphosphate Carboxylase.

Ribulose 1,5-diphosphate carboxylase has been purified from extracts of autotrophically grown Chlorella fusca by ammonium sulfate precipitation and centrifugation on a linear sucrose density gradient. The enzyme was homogeneous by the criterion of polyacrylamide gel electrophoresis. The molecular weight of the enzyme was 530,000, and it was composed of two types of subunit of molecular weight 53,000 and 14,000. Ribulose 1,5-diphosphate, CO(2), and Mg(2+) had Michaelis constant values of 15 mum, 0.3 mm, and 0.37 mm, respectively. At high bicarbonate concentration (17 mm and 50 mm), 6-phosphogluconate inhibited the enzyme, the inhibition being noncompetitive with respect to ribulose 1,5-diphosphate (Ki 0.065 mm), whereas at low bicarbonate concentration (1 mm), 6-phosphogluconate activated the enzyme. Oxygen was a competitive inhibitor with respect to CO(2), suggesting the enzyme also functions as an oxygenase. This was confirmed by direct assay, a 1: 1 stoichiometry between ribulose 1,5-diphosphate consumed and O(2) uptake being observed.

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PubMed1977

Penetration of Mannitol into the Intracellular Space of Chlorella sorokiniana.

It was found that in the alga, Chlorella sorokiniana, mannitol penetrated the plasmalemma (normally thought to be impermeable to mannitol) into the intracellular space. The rate of penetration is exponential and relatively slow, having a half-time of 6 to 12 minutes and requiring over 60 minutes for complete penetration. This penetration was demonstrated both by the Millipore filtration of cells incubated with (14)C-mannitol and by centrifugation of the cells through a silicon oil layer after incubation with tritiated water and (14)C-mannitol. Further, mannitol caused an inhibition of both autotrophic (on CO(2)) and heterotrophic (on glucose) growth. A low rate of mannitol metabolism was demonstrated, although this rate could not support heterotrophic growth.

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PubMed1978

Essential Sulfhydryl Group in the Transport-catalyzing Protein of the Hexose-Proton Cotransport System of Chlorella.

The polyene antibiotic nystatin transforms the sugar-proton contransport system of Chlorella to a mere facilitated diffusion system. This experimental condition was used to test the sugar-translocating unit of the active uptake system for possible essential sulfhydryl groups. It could be shown that the catalyzed translocation of sugar is sensitive to the sulfhydryl-reactive compound N-ethylmaleimide. Sugar flow by passive leak as induced by the detergent Triton X-100 is not affected by sulfhydryl reagents. These results show that the sugar-translocating carrier protein possesses a sulfhydryl group, which is essential for its function.

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PubMed1978

Physical and Kinetic Properties of the Nicotinamide Adenine Dinucleotide-specific Glutamate Dehydrogenase Purified from Chlorella sorokiniana.

The nicotinamide adenine dinucleotide-specific glutamate dehydrogenase (l-glutamate:NAD(+) oxidoreductase, EC 1.4.1.2) of Chlorella sorokiniana was purified 1,000-fold to electrophoretic homogeneity. The native enzyme was shown to have a molecular weight of 180,000 and to be composed of four identical subunits with a molecular weight of 45,000. The N-terminal amino acid was determined to be lysine. The pH optima for the aminating and deaminating reactions were approximately 8 and 9, respectively. The K(m) values for alpha-ketoglutarate, NADH, NH(4) (+), NAD(+), and l-glutamate were 2 mm, 0.15 mm, 40 mm, 0.15 mm, and 60 mm, respectively. Whereas the K(m) for alpha-ketoglutarate and l-glutamate increased 10-fold, 1 pH unit above or below the pH optima for the aminating or deaminating reactions, respectively, the K(m) values for NADH and NAD(+) were independent of change in pH from 7 to 9.6. By initial velocity, product inhibition, and equilibrium substrate exchange studies, the kinetic mechanism of enzyme was shown to be consistent with a bi uni uni uni ping-pong addition sequence. Although this kinetic mechanism differs from that reported for any other glutamate dehydrogenase, the chemical mechanism still appears to involve the formation of a Schiff base between alpha-ketoglutarate and an epsilon-amino group of a lysine residue in the enzyme. The physical, chemical, and kinetic properties of this enzyme differ greatly from those reported for the NH(4) (+)-inducible glutamate dehydrogenase in this organism.

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PubMed1978

Association of Potassium and Some Other Monovalent Cations with Occurrence of Polyphosphate Bodies in Chlorella pyrenoidosa.

Phosphate-starved Chlorella pyrenoidosa cells formed polyphosphate bodies (PB) upon transfer into nutrient solutions containing phosphate and potassium, or another monovalent cation, such as Na(+), NH(4) (+), Li(+), or Rb(+). The phenomenon was studied by chemical analyses, light microscopy, and electron microscopy.When the P-starved cells were transferred into a complete nutrient solution containing 100 micromolar P, they accumulated large quantities of P and K within several hours. The accumulation was accompanied by a corresponding appearance of PB in the cells. The absence of K from the medium prevented appreciable P accumulation and PB formation, but omitting Ca or Mg did not.The P-starved cells exposed to a simple solution of at least 20 micromolar H(3)PO(4) and 100 micromolar KHCO(3) responded in a similar manner as the cells exposed to the complete nutrient solution. However, the PB appeared structurally different.It is proposed that monovalent cations are essential for PB formation in C. pyrenoidosa. K is suggested to be a major component of PB formed in K-sufficient media.

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