Green Alga Chlorella Kessleri Research Articles

Diacylglyceryl-N,N,N-trimethylhomoserine-dependent lipid remodeling in a green alga, Chlorella kessleri

Membrane lipid remodeling contributes to the environmental acclimation of plants. In the green lineage, a betaine lipid, diacylglyceryl-N,N,N-trimethylhomoserine (DGTS), is included exclusively among green algae and nonflowering plants. Here, we show that the green alga Chlorella kessleri synthesizes DGTS under phosphorus-deficient conditions through the eukaryotic pathway via the ER. Simultaneously, phosphatidylcholine and phosphatidylethanolamine, which are similar to DGTS in their zwitterionic properties, are almost completely degraded to release 18.1% cellular phosphorus, and to provide diacylglycerol moieties for a part of DGTS synthesis. This lipid remodeling system that substitutes DGTS for extrachloroplast phospholipids to lower the P-quota operates through the expression induction of the BTA1 gene. Investigation of this lipid remodeling system is necessary in a wide range of lower green plants for a comprehensive understanding of their phosphorus deficiency acclimation strategies.

Different sensitivities of photosystem II in green algae and cyanobacteria to phenylurea and phenol-type herbicides: effect on electron donor side.

The effects of short-term treatment with phenylurea (DCMU, isoproturon) and phenol-type (ioxynil) herbicides on the green alga Chlorella kessleri and the cyanobacterium Synechocystis salina with different organizations of photosystem II (PSII) were investigated using pulse amplitude modulated (PAM) chlorophyll fluorescence and photosynthetic oxygen evolution measured by polarographic oxygen electrodes (Clark-type and Joliot-type). The photosynthetic oxygen evolution showed stronger inhibition than the PSII photochemistry. The effects of the studied herbicides on both algal and cyanobacterial cells decreased in the following order: DCMU>isoproturon>ioxynil. Furthermore, we observed that the number of blocked PSII centers increased significantly after DCMU treatment (204-250 times) and slightly after ioxynil treatment (19-35 times) in comparison with the control cells. This study suggests that the herbicides affect not only the acceptor side but also the donor side of PSII by modifications of the Mn cluster of the oxygen-evolving complex. We propose that one of the reasons for the different PSII inhibitions caused by herbicides is their influence, in different extents, on the kinetic parameters of the oxygen-evolving reactions (the initial S0-S1 state distribution, the number of blocked centers SB, the turnover time of Si states, misses and double hits). The relationship between the herbicide-induced inhibition and the changes in the kinetic parameters is discussed.

Gene cloning, sequence analysis, and expression profiles of a novel β-ring carotenoid hydroxylase gene from the photoheterotrophic green alga Chlorella kessleri.

In this study, a full-length complementary DNA (cDNA) sequence of β-ring carotenoid hydroxylase (CHY), designated Ckecyp97a1, was isolated via reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends (RACE) methods. The cloned Ckecyp97a1 cDNA was 2,264-bp in length, and contained an open reading frame (ORF) of 1,944-bp with 5'-terminal untranslated region (UTR) of 66-bp and 3'-terminal UTR of 254-bp and encoded a β-ring CHY protein of 647 amino acids. The deduced protein had a calculated molecular mass of 71.43kDa with an estimated isoelectric point (pI) of 6.72. Multiple sequence alignment and phylogenetic analysis revealed that Ckecyp97a1 was homologs to known chloroplastic cytochrome P450 (P450) CHY. The typical catalytic motifs of the P450 were highly conserved in the protein sequences of CkeCYP97A1. The Ckecyp97a1 transcriptional expression and carotenoids accumulation were observed under high light (HL) of different wavelengths (white: 390-770nm and blue: 420-500nm). The results revealed that Ckecyp97a1 transcript increased strongly throughout the course of the HL illumination treatment (22-70h) under white HL treatment, while decreased during 10-58h under blue HL treatment. The concentrations of lutein, α-carotene, and β-carotene were relatively steady and below the control level under both treatments. The zeaxanthin concentration was higher under white HL treatment than those under control and blue HL treatments. Ckecyp97a1 gene showed different expression patterns under different light wavelengths treatments. The data obtained in this study demonstrates that CkeCYP97A1 is the enzyme responsible for carotenoid hydroxylation involved in HL acclimation for photoheterotrophic green alga Chlorella kessleri CGMCC 4917.

Selective method for cyanobacterial bloom removal: hydraulic jet cavitation experience

The aim of this study was to investigate the suitability of hydraulic jet cavitation as a method for cyanobacterial water-bloom management. Effects of cavitation were studied on laboratory culture of the cyanobacterium Microcystis aeruginosa, on a culture of a green alga Chlorella kessleri (as a non-target species) as well as on a real cyanobacterial biomass with Microcystis sp. as a dominant species. Our results suggested that the cavitation treatment of cyanobacteria is capable of causing the disintegration of their gas vesicles. Using this treatment, up to 99 % removal efficiency of cyanobacteria was achieved. Moreover, no effect on cyanobacterial membrane integrity or metabolic activity was detected by flow cytometry; thus, hydraulic cavitation seems to be harmless from the viewpoint of possible release of cyanotoxins into the water column. The green algae (here C. kessleri) were not affected negatively by the cavitation, and thus, they may still act as the natural nutrient competitors of cyanobacteria in lakes, ponds or reservoirs treated by cavitation.

Rapid algal toxicity assay using variable chlorophyll fluorescence for Chlorella kessleri (chlorophyta)

Three methods of algal assays--the standard assay, microassay, and the proposed fluorescence assay--are compared from the point of view of reliability of EC50 detection, the minimum required time for the detection, sensitivity of individual measurement, i.e. at which cell density the particular assay can be used for EC50 estimation, and the time stability of the EC50 values. The assays were performed with green alga Chlorella kessleri strain LARG/1 growing in potassium dichromate solution in Z-medium ranging from 0.01 to 100 mg Cr L⁻¹. The inoculation cell density was set according to the standards to 10⁴ cells mL⁻¹ and according to spectrophotometer/plate reader detection limit. The average EC50 ranged from 0.096 to 0.649 mg Cr L⁻¹ and there were no significant differences in EC50 between the assay type and the inoculation methods with the exception of the significant difference between EC(c)50₇₂ (EC50 established from biomass measured as chlorophyll a concentration after 72 h of cultivation) in the standard assay and EC(r)50 (EC50 derived from growth rate) in the microassay in the standard inoculation experiment due to low variability of their values. The EC(f)50 (EC50 derived from variable fluorescence measurement) values correspond to EC50 values derived from the growth rates. Fluorescence measurement revealed the toxic effect of the chromium after 24 h of exposure at cell density of 5 x 10⁴ cells mL⁻¹, less by half than other used assay methods. The positive correlation of EC(f)50 and time was found in the standard inoculation experiment but opposite effect was observed at the spectrophotometric one.

High stability ferric chelates result in decreased iron uptake by the green alga Chlorella kessleri owing to decreased ferric reductase activity and chelation of ferrous iron

Cells of the green alga Chlorella kessleri Fott et Nováková use a reductive mechanism for iron acquisition. Iron-limited cells acquired iron more rapidly from a chelator with a lower stability constant for Fe3+ (hydroxyethylethylenediaminetriacetic acid (HEDTA)) than from a chelator with a higher stability constant (N,N′-di[2-hydroxybenzyl)ethylenediamine-N,N′-diacetic acid (HBED)). Furthermore, iron uptake rates decreased with increasing chelator concentrations at constant iron concentration. The negative effects of elevated HBED levels on iron uptake could be partly alleviated by the addition of Ga3+, which suggests that iron-free chelator has a negative effect on iron acquisition by competing for Fe2+ with the ferrous transport system. Furthermore, ferric reductase activity progressively decreased with increasing concentrations of both chelators (in the iron-free form). This effect was not alleviated by Ga3+ addition and was apparently caused by the direct inhibition of the reductase. Overall, we conclude that chelators with high stability constants for Fe3+ decrease iron acquisition rates by Strategy I organisms via three separate mechanisms.

Ferric and cupric reductase activities by iron‐limited cells of the green alga Chlorella kessleri: quantification via oxygen electrode

The colorimetric Fe2+ indicators bathophenanthroline disulfonic acid (BPDS) and 3-(2-pyridyl)-5,6-bis(4-phenylsulfonic acid)-1,2,4-triazine (FZ) are routinely used to assay for plasma membrane ferric reductase activity in iron-limited algal cells and also in roots from iron-limited plants. Ferric reductase assays using these colorimetric indicators must take into account the fact that Fe3+ chelators (e.g. ethylenediaminetetraacetic acid) can also in general bind Fe2+ and may therefore compete with the colorimetric Fe2+ indicators, leading to the potential for underestimation of the ferric reduction rate. Conversely, the presence of BPDS or FZ may also facilitate the reduction of Fe3+ chelates, potentially leading to overestimation of ferric reduction rates. Last, both BPDS and FZ have non-negligible affinities for Fe3+ in addition to their well-known affinities for Fe2+; this leads to potential difficulties in ascertaining whether free and/or chelated Fe3+ are potential substrates for the ferric reductase. Similar issues arise when assaying for cupric reductase activity using the colorimetric Cu+ indicator bathocuproinedisulfonic acid (BCDS). In this paper, we describe an oxygen-electrode-based assay (conducted in darkness) for both ferric and cupric reductase activities that does not use colorimetric indicators. Using this assay system, we show that the plasma membrane metal reductase activity of iron-limited cells of the green alga Chlorella kessleri reduced complexed Fe3+ (i.e. Fe3+ chelates) but did not reduce free (non-chelated) Fe3+, and also reduced free Cu2+ to Cu+, but did not reduce Cu2+ that was part of Cu2+ chelates. We suggest that the potential for reduction of free Fe3+ cannot be adequately assayed using colorimetric assays. As well, the BPDS-based assay system consistently yielded similar estimates of ferric reductase activity compared with the O2-electrode-based assays at relatively low Fe3+ concentration, but higher estimates at higher Fe3+ concentrations with chelators other than desferrioxamine mesylate. With respect to cupric reductase activity, the O2 electrode consistently provided much higher estimates; we suggest that this was as a result of Cu2+ chelation by BCDS leading to a large underestimation of the true cupric reduction rate. These results suggest that an O2-electrode-based metal reductase assay system has some specific advantages compared with the traditional colorimetric assay system, including especially the ability to discriminate between the reduction of free metal ions and chelated metal ions.

Toxicity of hexachlorobenzene and its transference from microalgae ( Chlorella kessleri) to crabs ( Chasmagnathus granulatus)

The aim of this work was to study the transference of hexachlorobenzene from a green alga ( Chlorella kessleri) to an estuary crab ( Chasmagnathus granulatus), and to analyze the toxic effects that the xenobiotic has on the latter. The effect of hexachlorobenzene uptake was evaluated measuring oxidative stress, Uroporphyrinogen decarboxylase activity and morphometric parameter alteration, and also performing a histological analysis of crab hepatopancreas. Results demonstrated that hexachlorobenzene enters the alga, is accumulated in it, and then transferred into the crab, causing a decrease in Uroporphyrinogen decarboxylase activity in both organisms. The high malondialdehyde levels detected in crab hepatopancreas after the toxic treatment suggested the existence of hexachlorobenzene-induced lipid peroxidation. Antioxidant defenses such as superoxide dismutase activity and reduced glutathione content fell below normal values on the fourth week of treatment. At the same time, the hepatosomatic index, used as a morphometric parameter, reduced 20% with respect to the control. The histological analysis revealed epithelium disorganization in hepatopancreas tubules, confirming the existence of structural damage caused by hexachlorobenzene.

Lipid Raft-Based Membrane Compartmentation of a Plant Transport Protein Expressed in Saccharomyces cerevisiae

The hexose-proton symporter HUP1 shows a spotty distribution in the plasma membrane of the green alga Chlorella kessleri. Chlorella cannot be transformed so far. To study the membrane localization of the HUP1 protein in detail, the symporter was fused to green fluorescent protein (GFP) and heterologously expressed in Saccharomyces cerevisiae and Schizosaccharomyces pombe. In these organisms, the HUP1 protein has previously been shown to be fully active. The GFP fusion protein was exclusively targeted to the plasma membranes of both types of fungal cells. In S. cerevisiae, it was distributed nonhomogenously and concentrated in spots resembling the patchy appearance observed previously for endogenous H(+) symporters. It is documented that the Chlorella protein colocalizes with yeast proteins that are concentrated in 300-nm raft-based membrane compartments. On the other hand, it is completely excluded from the raft compartment housing the yeast H(+)/ATPase. As judged by their solubilities in Triton X-100, the HUP1 protein extracted from Chlorella and the GFP fusion protein extracted from S. cerevisiae are detergent-resistant raft proteins. S. cerevisiae mutants lacking the typical raft lipids ergosterol and sphingolipids showed a homogenous distribution of HUP1-GFP within the plasma membrane. In an ergosterol synthesis (erg6) mutant, the rate of glucose uptake was reduced to less than one-third that of corresponding wild-type cells. In S. pombe, the sterol-rich plasma membrane domains can be stained in vivo with filipin. Chlorella HUP1-GFP accumulated exactly in these domains. Altogether, it is demonstrated here that a plant membrane protein has the property of being concentrated in specific raft-based membrane compartments and that the information for its raft association is retained between even distantly related organisms.

Differences between two green algae in biological availability of iron bound to strong chelators

N,N′-di(2-hydroxybenzoyl)-ethylenediamine-N,N′-diacetic acid (HBED) is a very strong Fe3+ chelator. Strategy I vascular plants, which use a reductive system for iron acquisition, similar to many green algae, are able to access iron from HBED (R.L. Chaney. 1988. J. Plant Nutr. 11: 1033–1050). However, iron-limited cells of the Strategy I green alga Chlamydomonas reinhardtii Dangeard were unable to access iron present as Fe3+–HBED. In contrast, Fe3+ chelated with hydroxyethylethylenediaminetriacetic acid (HEDTA; a weaker chelator) was rapidly taken up by iron-limited Chlamydomonas cells. Chlamydomonas ferric reduction rates with Fe3+–HBED were approximately 15% of the rate observed with Fe3+–HEDTA, suggesting that low reduction rates with Fe3+–HBED might be one factor in the low rate of iron acquisition. By contrast, iron-limited cells of the Strategy I green alga Chlorella kessleri Fott et Nováková were able to rapidly assimilate Fe3+ chelated by HBED, although ferric reduction rates with Fe3+–HBED were approximately 38% the rate of activity with Fe3+–HEDTA. Similar differential iron uptake rates for the two algal species were obtained using the strong Fe3+ chelator (and siderophore analogue) desferrioxamine B mesylate and the cyanobacterial siderophore schizokinen. These results suggest that there are differences among Strategy I green algae in their abilities to acquire Fe3+ from various ferric chelates: not all Strategy I algae can equally access tightly complexed Fe3+. Chlamydomonas appears to be the first documented Strategy I organism that is unable to acquire iron from Fe3+–HBED. These results also suggest that green algal iron acquisition from siderophores is species dependent. Finally, we suggest that iron acquisition from Fe3+–HBED might serve as an assay for an organisms’ ability to access tightly complexed iron.

Identification of very long chain fatty acids by atmospheric pressure chemical ionization liquid chromatography‐mass spectroscopy from green alga Chlorella kessleri

AbstractA method is described for the enrichment of very long chain fatty acids (VLCFAs) from total fatty acids of heterotrophically cultivated green freshwater alga Chlorella kessleri and their identification as picolinyl esters by means of liquid chromatography‐mass spectrometry with atmospheric pressure chemical ionization (LC‐MS with APCI). The method is based on the use of preparative reversed phase HPLC of hundred‐milligram amounts and their subsequent identification by microbore APCI LC‐MS. A combination of these two techniques was used to identify unusual VLCFAs up to C47, both saturated and monounsaturated, with two positional isomers (ω‐9 and ω‐26).

OXYGEN CONSUMPTION ASSOCIATED WITH FERRIC REDUCTASE ACTIVITY AND IRON UPTAKE BY IRON‐LIMITED CELLS OF CHLORELLA KESSLERI (CHLOROPHYCEAE)

Plasma membrane ferric reductase activity was enhanced 5‐fold under iron limitation in the unicellular green alga Chlorella kessleri Fott et Nováková. Furthermore, ferric reductase activity in iron‐limited cells was approximately 50% higher in the light than in the dark. In contrast, iron uptake rates of iron‐limited cells were unaffected by light versus dark treatments. Rates of iron uptake were much lower than rates of ferric reduction, averaging approximately 2% of the dark ferric reduction rate. Ferric reduction was associated with an increased rate of O2 consumption in both light and dark, the increase in the light being approximately 1.5 times as large as in the dark. The increased rate of O2 consumption could be decreased by half by the addition of catalase, indicating that H2O2 is the product of the O2 consumption and that the increased O2 consumption is nonrespiratory. The stimulation of O2 consumption was almost completely abolished by the addition of bathophenanthroline disulfonate, a strong chelator of Fe2+. Anaerobic conditions or the presence of exogenous superoxide dismutase affected neither ferric reduction nor iron uptake. We suggest that the O2 consumption associated with ferric reductase activity resulted from superoxide formation from the aerobic oxidation of Fe2+, which is the product of ferric reductase activity. At saturating concentrations of Fe3+ chelates, ferric reductase activity is much greater than the iron uptake rate, leading to rapid oxidation of Fe2+ and superoxide generation. Therefore, O2 consumption is not an integral part of the iron assimilation process.

Active transport of CO 2 and bicarbonate is induced in response to external CO 2 concentration in the green alga Chlorella kessleri

Active transport of CO 2 and bicarbonate is induced in response to external CO 2 concentration in the green alga Chlorella kessleri

Active transport of CO2 and bicarbonate is induced in response to external CO2 concentration in the green alga Chlorella kessleri

The time-course of induction of CO(2) and HCO(3)- transport has been investigated during the acclimation of high CO(2)-grown Chlorella kessleri cells to dissolved inorganic carbon (DIC)-limited conditions. The rate of photosynthesis of the cells in excess of the uncatalysed supply rate of CO(2) from HCO(3)- was taken as an indicator of HCO(3)- transport, while a stimulation of photosynthesis on the addition of bovine carbonic anhydrase was used as an indicator of CO(2) transport. The maximum rate of photosynthesis (Pmax) was similar for high CO(2)-grown and low CO(2)-grown cells, but the apparent whole cell affinity for DIC and CO(2) of high CO(2)-grown cells was found to be about 30-fold greater than in air-grown cells, which indicates a lower affinity for DIC and CO(2). It was found that HCO(3)- and CO(2) transport were induced in 5.5 h in cells acclimating to air in the light and in the presence and absence of 21% O(2), which indicates that a change in the CO(2)/O(2) ratio in the acclimating medium does not trigger induction of DIC transport. No active DIC transport was detected in high CO(2)-grown cells maintained on high CO(2) for 5.5 h in the presence of 5 mM aminooxyacetate, an aminotransferase inhibitor. These results indicate no involvement of photorespiration in triggering induction. Active DIC transport induction was inhibited in cells treated with 5 microgram ml(-1) cycloheximide, but was unaffected by chloramphenicol treatment, indicating that the induction process requires de novo cytoplasmic protein synthesis. The total DIC concentration eliciting the induction and repression of CO(2) and HCO(3)- transport was higher at pH 7.5 than at pH 6.6. The concentrations of external CO(2) required for the induction and repression of DIC transport were 0 and 120 microM, respectively, and was independent of the pH of the acclimation medium. Prolonged exposure to a critical external CO(2) concentration elicits the induction of DIC transport in C. kessleri.

Subcellular Localization of the Inducible Chlorella HUP1 Monosaccharide-H+ Symporter and Cloning of a Co-Induced Galactose-H+ Symporter

The unicellular green alga Chlorella kessleri can induce monosaccharide-H+ symport catalyzing the energy-dependent transport of D-glucose (D-Glc) and several other pentoses and hexoses across the plasmalemma. The gene coding for the inducible HUP1 monosaccharide-H+ symporter has been cloned and the protein has been characterized previously. The data presented in this paper demonstrate that the presence of the HUP1 gene product alone is not sufficient to cover the broad substrate specificity of monosaccharide transport in induced Chlorella cells. Two other HUP genes are shown to be co-induced in Chlorella in response to D-Glc in the medium. The cloning of HUP2 and HUP3 cDNA and genomic sequences is described, both being very homologous to HUP1. Modification of the 5' untranslated sequences of full-length cDNA clones of HUP2 and HUP3 allowed the functional expression of both transporters in Schizosaccharomyces pombe. HUP2 was shown to be a galactose-H+ symporter, whereas the substrate specificity of the HUP3 gene product is very similar to that of the HUP1 protein. However, HUP3 does not seem to be induced to high levels in Glc-treated Chlorella cells. Results are also presented proving that the product of the HUP1 gene is localized in the plasmalemma of D-Glc-induced Chlorella cells and is absent in plasma membranes of noninduced cells. Incubation of thin sections of Chlorella cells with anti-HUP1 antibodies and a fluorescence-labeled, second antibody yielded a ring of fluorescence on the surface of Glc-induced Chlorella cells.

Effect of atrazine herbicide on growth, photosynthesis, protein synthesis, and fatty acid composition in the unicellular green alga Chlorella kessleri

Sublethal atrazine concentrations induced a general inhibition on growth, photosynthesis, and dark respiration in the green alga Chlorella kessleri. 14 C-protein hydrolysate indicated a maximum incorporation level by 5 μ M atrazine after 48 hr. Progressive reduction in protein synthesis was associated with increasing herbicide concentration at all experimental periods. The herbicide had preferential effect on the associated fatty acid composition of total and phospholipids. After 24 and 48 hr the herbicide stimulated fatty acids synthesis at concentrations where photosynthesis was inhibited. Meanwhile, stearic and miristic acids disappeared at 15 μ M after 24 hr and the total polyunsaturated fatty acids were not affected after 48 hr. Fatty acid synthesis was sensitive to treatment at 72 hr by 5 and 10 μ M atrazine, whereas the total saturated fatty acids were completely inhibited.

Response of fresh water phytoplanktonic algae Chlorella Kessleri and Synechocystis PCC 6803 to anthelmintic activity of the wild egyptian plant Calendula micrantha officinalis

Calendula micrantha water extract was added to cultures of the green alga Chlorella kessleri and cyanobacterium Synechocystis PCC 6803. Treatments enhanced growth of the cultures, increased chlorophyll content, and 14C-labeled protein synthesis. However, a general induced protein synthesis pattern by 0.5% extract was observed reaching maximum level after 24 and 36 h in Chlorella and Synechocystis, respectively. Oxygen evolution in Chlorella was markedly inhibited after 12 h and slightly inhibited at longer exposure times and accompanied by a two- to fourfold increase in respiration at all experimental periods. Extracts induced oxygen evolution after 48 h in Synechocystis reaching maximum (52%) at 0.5%. The cyanobacterium exhibited a general reduced respiration pattern after 24 h which slightly increased afterwards. Calendula extract at 0.7% after 12 h had a preferential effect on both unicultures, as in Chlorella it markedly reduced protein synthesis and the oxygen evolution was minimum, whereas in Synechocystis it slightly reduced the level of protein synthesis with increasing oxygen evolution. The preferential effect was exhibited by all extract concentrations after 48 h, where protein synthesis was moderately induced in Chlorella and markedly inhibited in Synechocystis.

A direct measurement of thermal energy dissipation in the photosynthetic apparatus during induction of fluorescence

A calorimetric method was applied to monitor the heat emission during the induction of photosynthesis (fluorescence) in the dark-adapted green alga Chlorella kessleri suddenly illuminated with HeNe laser light. The same method was also applied to record the heat emission from an illuminated preparation of the major light-harvesting pigment-protein complex of photosystem II (LHCII). The results are discussed in terms of the kinetics of recorded fluorescence changes. The direct measurement of thermal energy dissipation by the calorimetric technique is compared with indirect methods based on photothermal techniques such as photoacoustic spectroscopy.

Analysis of sterol esters from alga and yeast by high-performance liquid chromatography and capillary gas chromatography-mass spectrometry with chemical ionization

Sterol esters from the green alga Chlorella kessleri and the yeast Saccharomyces cerevisiae were analysed by tandem high-performance liquid chromatography (HPLC-HPLC). Non-polar lipids were separated by normal-phase HPLC into individual classes and the fraction of sterol esters was subsequently separated by reversed-phase HPLC into intact molecular species. Further separation and identification of the fractions after HPLC were effected by capillary gas chromatography-mass spectrometry (cGC-MS) with positive and/or negative chemical ionization. A technique consisting of direct injection of the sample after its passage through the detector into the on-column injector of the gas chromatograph was used to transfer the sample from HPLC-HPLC to cGC-MS. By means of this method it was possible to demonstrate more than 30 sterols in both the alga and the yeast, and more than 20 new sterol esters were detected.

Oligomeric Forms Of Pyruvate Kinase From Chlorella With Different Kinetic Properties

Fast protein liquid chromatography on Superose 6 of crude extracts from the chlorophyllfree mutant no. 20 of the unicellular green alga Chlorella kessleri reveals two possibly oligomeric forms of pyruvate kinase (2.7.1.40). Their occurrence is markedly altered in the course of heterotrophic growth with changing levels of exogenous glucose as carbon source with only one enzyme species with a MW of 400 kDa existing in growing cells, two forms of 400 and 580 kDa in resting cells. Substrate affinity towards PEP of the 400 kDa form is better than that of the 580 kDa species; responses to the effector AMP are different as well. In vitro, addition of PEP or of AMP leads to the formation of higher MW enzyme species with MW of 730, 1050 and 1400 kDA without affecting the total activity. In vivo alterations in the levels of several metabolites including PEP upon addition of glucose have been shown to occur. Therefore, it is discussed, whether changes in the concentration of intermediates and effectors may provide the mechanism for the increased rate of carbohydrate degradation by affecting the occurrence and/or ratio of the various PK forms with different kinetic and regulatory properties. Upon blue light irradiation, which also stimulates carbohydrate breakdown of the Chlorella mutant cells, the distribution of PK is shifted towards the species with higher substrate affinity, a result being in accordance with the above conception