Glycerolipid synthesis in Chlorella kessleri 11h: I. Existence of a eukaryotic pathway

The fatty acid distributions at the sn-1 and sn-2 positions in major chloroplast lipids of Chlorella kessleri 11h, monogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG), were determined to show the coexistence of both C₁₆ and C₁₈ acids at the sn-2 position, i.e. of prokaryotic and eukaryotic types in these galactolipids. For investigation of the biosynthetic pathway for glycerolipids in C. kessleri 11h, cells were fed with [¹⁴C]acetate for 30 min, and then the distribution of the radioactivity among glycerolipids and their constituent fatty acids during the subsequent chase period was determined. MGDG and DGDG were labeled predominantly as the sn-1-C₁₈-sn-2-C₁₆ (C₁₈/C₁₆) species as early as by the start of the chase, which suggested the synthesis of these lipids within chloroplasts via a prokaryotic pathway. On the other hand, the sn-1-C₁₈-sn-2-C₁₈ (C₁₈/C₁₈) species of these galactolipids gradually gained radioactivity at later times, concomitant with a decrease in the radioactivity of the C₁₈/C₁₈ species of phosphatidylcholine (PC). The change at later times can be explained by the conversion of the C₁₈/C₁₈ species of PC into galactolipids through a eukaryotic pathway. The results showed that C. kessleri 11h, distinct from most of other green algal species that were postulated mainly to use a prokaryotic pathway for the synthesis of chloroplast lipids, is similar to a group of higher plants designated as 16:3 plants in terms of the cooperation of prokaryotic and eukaryotic pathways to synthesize chloroplast lipids. We propose that the physiological function of the eukaryotic pathway in C. kessleri 11h is to supply chloroplast membranes with 18:3/18:3-MGDG for their functioning, and that the acquisition of a eukaryotic pathway by green algae was favorable for evolution into land plants.     

Expression of eukaryotic plasma membrane transporter HUP1 from Chlorella kessleri in Escherichia coli

To study the effect of sterols on the activity of the eukaryotic plasma membrane transporter, the hexose-proton symporter HUP1 from the unicellular alga Chlorella kessleri was expressed in Escherichia coli, a prokaryotic microorganism containing virtually no sterols. Under certain conditions, the recombinant protein was partially active in this prokaryotic organism. The heterologously produced HUP1p was purified from membrane fractions of E. coli and reconstituted in an in vitro system. The presence of ergosterol during solubilization, purification and reconstitution resulted in an increased activity of the reconstituted protein. Its activity, however, was 5-6 times lower as compared to the activity of HUP1p produced in Saccharomyces cerevisiae membranes and solubilized, purified, and reconstituted under the same conditions as above.     

Effect of Temperature on Starch Degradation in Chlorella vulgaris 11h Cells

Analysis of products formed in Chlorella vulgaris 11 h cellsduring photosynthesis in air containing 3,000 ppm ¹⁴CO₂ at varioustemperatures revealed that the level of ¹⁴C-starch was maximumaround 20–24?C and decreased with further rise in temperatureuntil 40?C, while ¹⁴C-sucrose greatly increased at temperaturesabove about 28?C. Elevating the temperature from 20 to 38?Cduring photosynthetic ¹⁴CO₂ fixation resulted in a remarkabledecrease in ¹⁴C in starch and a concomitant increase in ¹⁴Cin sucrose. This conversion of starch to sucrose when shiftingthe temperature from 20 to 38?C proceeded even in the dark.Hydrolysis of sucrose by rß-fructosidase showed that,irrespective of the experimental conditions, the radioactivitiesin sucrose were equally distributed between glucose and fructose.The enhancement of starch degradation with temperature risewas more remarkable than that of the activity of ribulose bisphosphatecarboxylase from the same cells. When Chlorella cells whichhad been preloaded with ¹⁴C-starch after photosynthesis for30 min at 20?C were incubated in the dark for an additional30 min at 20?C, ¹⁴C-starch was degraded by only about 4%. However,the values after 30-min dark incubation at 28, 32, 36 and 40?Cwere increased by about 10, 19, 36 and 50%, respectively. Duringthe temperature-dependent conversion of starch to sucrose, nosignificant amount of radioactivity accumulated in free glucoseand maltose. (Received October 27, 1981; Accepted January 9, 1982)     

Form of inorganic carbon utilized for photosynthesis in Chlorella vulgaris 11 h cells

The rate of photosynthetic ¹⁴CO₂ fixation in Chlorella vulgaris11h cells in the presence of 0.55 mM NaH¹⁴CO₃ at pH 8.0 (20?C)was greatly enhanced by the addition of carbonic anhydrase (CA).However, when air containing 400 ppm ¹⁴CO₂ was bubbled throughthe algal suspension, the rate of ¹⁴CO₂ fixation immediatelyafter the start of the bubbling was suppressed by CA. Theseeffects of CA were observed in cells which had been grown inair containing 2% CO₂ (high-CO₂ cells) as well as those grownin ordinary air (containing 0.04% CO₂, low-CO₂ cells). We thereforeconcluded that, irrespective of the CO₂ concentration givento the algal cells during growth, the active species of inorganiccarbon absorbed by Chlorella cells is free CO₂ and they cannotutilize bicarbonate. The effects observed in the high-CO₂ cellswere much more pronounced than those in the high-CO₂ cells.This difference was accounted for by the difference in the affinityfor CO₂ in photosynthesis between the high- and low-CO₂ cells. (Received May 19, 1978; )     

Influence of red light on the activity of phosphofructokinase in Chlorella kessleri

In crude extracts of the unicellular green alga Chlorella kessleri Fott et Novákóva grown in red light the activity of the glycolytic enzyme phosphofructokinase (PFK, EC 2.7.1.11) is about 40% higher compared to white light conditions giving the same dry matter production. Application of cycloheximide and density labelling with D₂O indicate that this increase depends on the de novo synthesis of the enzyme: Twelve h of illumination at a fluence rate of 7 × 10¹⁸ quanta m⁻² s⁻¹ (11.6 μmol m⁻² s⁻¹) suffice to saturate the effect. In autotrophically grown algae maximal increase in enzyme saturate the effect. In autotrophically grown algae maximal increase in enzyme activity is reached in light of 680 nm, while in 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU)-poisoned, glucose-fed cells, light of wavelengths around 727 nm is most effective. Involvement of a phytochrome-like photoreceptor is discussed.     

Adaptation in a eukaryotic pathway

Comment on: Takeda K, et al. Sci Signal 2012; 5:2     

Influence of blue light on the activity of phosphofructokinase in Chlorella kessleri

In crude extracts of Chlorella kessleri Fott and Novákóva cells grown autotrophically in white light the activity of phosphofructokinase (PFK, EC 2.7.1.11) is 62.9 ± 1.5 nmol (mg protein)⁻¹ min⁻¹ under optimized test conditions. It is greatly increased in red [88.3 ± 1.8 nmol (mg protein)⁻¹ min⁻¹], but somewhat decreased [57.0 ± 0.5 nmol (mg protein)⁻¹ min⁻¹] in blue light of equal productivity. Mixtures of blue and red light yield the low activity as long as blue light represents at least 35% of the total quantum fluence rate. The rough wavelength dependence of the counteracting effect of short wavelength light on the increasing effect of red light exhibits a broad peak at 460 nm, reminiscent of action spectra of the blue/UV photoreceptors(s). Upon transfer of red light-grown cells to blue light, the decrease develops slowly within 72 h; it cannot be prevented by 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU). Since there is less carbohydrate in blue than in red light-exposed cells, correlations between biosynthesis of PFK and level of carbohydrate are discussed, based on the assumption that red light decreases and/or blue light increases the transport of metabolites across the chloroplast envelope.     

Glycerolipid biosynthesis in rat adipose tissue. Effect of polyamines on triglyceride synthesis.

Triacylglycerol formation from sn-glycerol 3-phosphate and 1,2-diacyl-sn-glycerol was markedly elevated in the presence of spermine and spermidine. This was attributed to the activation of microsomal sn-glycerol 3-phosphate acyltransferase and 1,2-diacyl-sn-glycerol acyltransferase and to the inhibition of palmitoyl-CoA hydrolase. Spermine was more effective than spermidine, and putrescine did not stimulate triacylglycerol formation. The stimulatory effect of spermine on triacylglycerol-forming enzymes was observed in the presence of Mg²⁺ and was apparent in the presence or absence of bovine serum albumin. The activation of 1,2-diacyl-sn-glycerol acyltransferase by spermine was specific, and other diacylglycerol-utilizing enzymes were not affected under these conditions. These studies demonstrate that polyamines may be important regulators of triacylglycerol formation in adipose tissue.     

Glycerolipid synthesis in isolated adipocytes: substrate dependence and influence of norepinephrine.

Studies of fatty acid (FA) esterification by adipocytes have led to conflicting views with respect to how the process is regulated by norepinephrine (NE). It remains unclear whether NE directly modulates the pathway or whether its effects are indirect and reflect its well-known action to activate lipolysis. Changes in lipolysis can complicate estimation of esterification rates by altering both medium FA and the hydrolysis of newly formed FA esters. In this report, we describe an experimental approach that determined the effect of NE on FA esterification, amidst the complications introduced by activation of lipolysis. Esterification rates were estimated from the simultaneous incorporations (0.1-60 min) of [14C]glucose and [3H]oleate into diglyceride (DG), phospholipid (PL), and triglyceride (TG). Saturation kinetics of incorporation rates, with respect to FA, and more specifically to unbound or albumin-free FA (ubFA), were determined in both basal and NE-treated cells. To obtain true estimates of ester synthesis, incorporation rates were adjusted for label loss from breakdown of labeled esters. Our findings were: 1) In basal versus NE-treated cells, [3H]oleate, on its pathway to esterification, was diluted, respectively, by 2 and 50% of measured cell FA, and the diluting FA appeared derived from lipolysis. 2) Syntheses of PL, DG, and TG, estimated from incorporation of [14C]glucose, saturated at low ubFA. The Km for TG synthesis (0.06 microM) was within the physiological range of ubFA which meant that changes in plasma FA will modulate TG synthesis. PL synthesis, on the other hand (Km less than 0.01 microM), would be largely saturated under physiological conditions. 3) NE treatment increased the molar ratio of FA to albumin in the medium an average 8-fold and ubFA about 87-fold. In addition, NE accelerated hydrolysis of labeled PL and DG. Adjusting incorporation rates for these changes indicated that NE does not directly regulate glyceride synthesis. The assays described should allow estimation of glycerolipid synthesis under various metabolic or disease states and will distinguish direct effects from those reflecting changes in FA concentration or in hydrolysis of labeled FA esters.     

Glycerolipid synthesis in rat adipose tissue. II. Properties and distribution of phosphatidate phosphatase

The properties and subcellular distribution of phosphatidate phosphatase (EC 3.1.3.4) from adipose tissue have been investigated. The enzyme was assayed using both aqueous phosphatidate and membrane-bound phosphatidate as substrates. When measured with aqueous substrate, activity was detected in the mitochondria, the microsomes, and the soluble fraction. Mg(2+) at low concentration stimulated the phosphatidate phosphatase from soluble and microsomal fractions but had no effect on the mitochondrial phosphatidate phosphatase. At higher concentration Mg(2+) was inhibitory. In the presence of Mg(2+), the phosphatidate phosphatase from soluble and microsomal fractions was active against membrane-bound phosphatidate. No activity was demonstrated with membrane-bound substrate in the absence of Mg(2+). Mitochondria did not contain activity toward the membrane-bound substrate. The rate of utilization of aqueous phosphatidate was always higher than that of membrane-bound substrate. These results indicate that there are at least two different phosphatidate phosphatases in adipose tissue.     

Blue and Red Light-dependent Alterations in the Ratio of two Forms of Glutamine Synthetase in Chlorella kessleri

Two forms of glutamine synthetase (EC 6.3.1.2) can be separated in crude extracts of Chlorella kessleri on the basis of their different surface charges. The two enzyme forms (GS1 and GS2) respond differently upon transferring the cells from darkness to autotrophic growth in white light: the activity of GS2 increases, that of GS1 remains unchanged. The increase in GS2 activity is only brought about by blue light; in red light GS2 activity appears to be uninfluenced, while that of GS1 increases. There are no indications of wavelength-dependent oligomerization processes as a cause for the observed activity alterations. There is however, a strong influence of inhibitors of protein biosynthesis. Cycloheximide and lincomycin both affect the blue light-dependent increase in activity of GS2, cycloheximide preventing that of GS1 in red lgiht completely. Since literature data point to localization of GS2 in the chloroplast, and GS1 in the cytosol, the data are discussed in view of two different photoreceptors involved in the regulation of the amounts of GS1 and GS2 in different compartments of the Chlorella cell.     

Transport and Fixation of Inorganic Carbon in the Cells of Chlorella vulgaris 11h Grown under Ordinary Air

Intracellular accumulation of inorganic carbon (C_i) and itsfixation in photosynthesis were investigated using siliconeoil layer filtering centrifugation technique with the cellsof Chlorella vulgaris 11h grown under ordinary air. Both CO₂and HCO₃^– were transported into the cells from the reactionmedium and accumulated in the cells, but the rate of transportwas much faster for the former than the latter. ¹⁴C-fixationfrom the total transported C_i was much more efficient when CO₂was added in the external medium than when HCO₃^– was added.This indicates that CO₂ and HCO₃^– were not converted tothe common compound in the cells during the initial period ofphotosynthesis. Accumulation of Cⁱ into the cells was much lesssusceptible to low temperature than its fixation. Accumulationof Cⁱ was also observed in the dark. Ethoxyzolamide, an inhibitorof carbonic anhydrase (CA), inhibited the fixation of accumulatedCO₂ in the cells, suggesting that CA enhanced the supply ofCO₂ to the reaction site of ribulose bisphosphate carboxylasein the stroma. Mechanism for transport and fixation of Cⁱ duringphotosynthesis in low-CO₂ cells of C. vulgaris 1lh was proposedfrom these results. (Received March 19, 1986; Accepted June 26, 1986)     

Evidence for a new biologic pathway of androstenedione synthesis from 11-deoxycortisol

17-Hydroxyprogesterone is a well-known precursor of androstenedione in adrenal biosynthesis. This study using sheep adrenal incubations demonstrates that 11-deoxycortisol, the precursor of cortisol synthesis, also can be a precursor of androstenedione. Indeed, our data show that androstenedione synthesis is negatively correlated to the synthesis of cortisol and cortisone. This fact allowed us to infer that this new pathway is closely related to the activity of the 11 beta-hydroxylase that is responsible for the synthesis of cortisol. Indeed, when the activity of this enzyme is impaired, 11-deoxycortisol follows the pathway that leads to androstenedione synthesis in the adrenals. This pathway could explain, at least in part, the marked increase of androstenedione observed in congenital adrenal hyperplasia due to 11 beta-hydroxylase deficiency.     

Factors Controlling Induction of Carbonic Anhydrase in Chlorella vulgaris 11h: Effects of CO2 and O2

Increase of carbonic anhydrase activity was enhanced by decreasingthe O₂ concentration when Chlorella vulgaris 11h cells grownunder 3% CO₂2 in ordinary air were transferred to low CO₂ conditions.The carbonic anhydrase activity finally attained under the steadystate was dependent on the CO₂ concentration, irrespective ofthe O₂ concentration used. (Received April 24, 1988; Accepted February 23, 1988)     

Role of subunits in eukaryotic Photosystem I.

Photosystem I (PSI) of eukaryotes has a number of features that distinguishes it from PSI of cyanobacteria. In plants, the PSI core has three subunits that are not found in cyanobacterial PSI. The remaining 11 subunits of the core are conserved but several of the subunits have a different role in eukaryotic PSI. A distinguishing feature of eukaryotic PSI is the membrane-imbedded peripheral antenna. Light-harvesting complex I is composed of four different subunits and is specific for PSI. Light-harvesting complex II can be associated with both PSI and PSII. Several of the core subunits interact with the peripheral antenna proteins and are important for proper function of the peripheral antenna. The review describes the role of the different subunits in eukaryotic PSI. The emphasis is on features that are different from cyanobacterial PSI.     

Glycerolipid biosynthesis in rat adipose tissue. 11. Effects of polyamines on Mg2+-dependent phosphatidate phosphohydrolase

The effect of polyamines (spermine, spermidine and putrescine) on the Mg2+-dependent phosphatidate phosphohydrolase was investigated. Phosphatidate phosphohydrolase activity was measured in the presence of aqueous dispersed phosphatidate as substrate, and the release of inorganic phosphate was taken as a measure of phosphatidate phosphohydrolase activity. In the presence of various polyamines there was activation of the Mg2+-dependent phosphatidate phosphohydrolase activity. Under this condition, the Km of enzyme towards phosphatidase decreased from 1.6 x 10(-4) to 9.8 x 10(-5) M and the Mg2+ requirement decreased from 5 to 0.5 mM. These polyvalent cations did not replace Mg2+, but potentiate the phosphohydrolase activity in the presence of Mg2+. The activation of Mg2+-dependent phosphatidate phosphohydrolase activity by polyamines was observed in the presence of 3-sn-phosphatidylcholine, suggesting that these modulators of phosphatidate phosphohydrolase activity may be acting through different mechanisms. These studies demonstrate that polyamines may be important regulators of Mg2+-dependent phosphatidate phosphohydrolase activity in adipose tissue.     

Nucleotide regulation of a eukaryotic protein synthesis initiation complex;.

Formation of a ternary initiation complex containing Met-tRNAf, GTP and eukaryotic initiation factor 2, is the first step in sequential assembly of the initiation complex. The concentration of GTP required for half maximal formation of the ternary complex is 2.5 with 10(-6) M. GDP is a potent competitive inhibitor of ternary complex formation with Ki = 3.4 with 10(-7) M. The nucleotide binding site on eukaryotic initiation factor 2 demonstrates relative specificity for GDP with KD(GDP) = 3.0 with 10(-8) M; 100-fold higher concentrations of GTP than GDP are required for displacement of either [(3)H]GDP or [(3)h]gtp from the necleotide binding site. An ATP-dependent stimulation of ternary complex formation observed in partially purified initiation factor preparations is due to nucleoside diphosphate kinase (EC 2.7.4.6) which serves to remove inhibitory levels of GDP by phosphorylation with ATP. Since GTP is hydrolyzed to GDP during protein synthesis, this provides a mechanism by which the ATP:ADP ratio may regulate the rate of initiation of protein synthesis.