Messenger RNA for isocitrate lyase from Chlorella fusca var. vacuolata

Chlorella fusca cultures growing in the light and adapting to acetate in the dark were labelled with adenine-³H and adenine-¹⁴C, respectively. Poly(A)-containing RNA from the mixed cultures was analysed for ¹⁴C/³H ratio after polyacrylamide gel electrophoresis in 98% formamide. The RNA from acetateadapting C. fusca cells contained excess label migrating in the gels at a position equivalent to about 0.85×10⁶ mol.wt. Partially purified anti-isocitrate lyase serum linked to p-aminobenzoyl-cellulose bound 3.5–13% of polysomes from acetate-adapting C. fusca, containing 5–10% of polysomal poly(A)-containing RNA. The antibody-bound poly(A)-containing RNA fraction showed a unimodal size distribution with a mean size of about 0.85×10⁶ mol.wt. after electrophoresis on 4% polyacrylamide gels in 98% formamide. Cell-free translation assays showed a three-fold enrichment of isocitrate lyase mRNA after antibody selection of polysomes and indicated that isocitrate lyase mRNA was abundant in acetate-adapting C. fusca cells.Abbreviations A ₂₆₀ unit The amount of material in 1.0 ml giving an absorbance of 1.0 at 260 nm in a 1 cm light path - PAB-cellulose p-aminobenzoyl-cellulose - SDS sodium lauryl sulphate To whom offprint requests are to be sent     

Catabolite inactivation of isocitrate lyase from Saccharomyces cerevisiae

A reversible carbon catabolite inactivation step is described for isocitrate lyase from Saccharomyces cerevisiae. This reversible inactivation step of isocitrate lyase is similar to that described for fructose 1,6-bisphosphatase. Addition of 2,4-dinitrophenol, nystatin or glucose to cultures, grown in ethanol as carbon source, caused a rapid loss of the isocitrate lyase and fructose 1,6-bisphosphatase activities at pH 5.5 but not at pH 7.5. These results suggest that intracellular acidification and thus a cAMP increase is involved in the catabolite inactivation mechanism of both enzymes. From results obtained by addition of glucose to yeast cultures at pH 7.5 it was concluded that others factors than cAMP can play a role in the catabolite inactivation mechanism of both enzymes.     

Inhibition of Candida albicans isocitrate lyase activity by cadiolides and synoilides from the ascidian Synoicum sp.

Tris-aromatic furanones (1?4) and related bis-aromatic diesters (5 and 6) isolated from the dark red ascidian Synoicum sp., were evaluated for their inhibitory activities toward Candida albicans isocitrate lyase (ICL). These studies led to the identification of compounds 1, 3, and 4 as potent ICL inhibitors, with IC₅₀ values of 7.62, 17.16, and 10.36 μM, respectively. Growth phenotype of ICL deletion mutants and Northern blot analysis data indicated that compound 1 inhibits the ICL expression in C. albicans under C₂ carbon utilizing condition.     

Embryonic control of isocitrate lyase activity in cotyledons of germinating soybean

The activity of isocitrate lyase (EC 4.1.3.1) in the cotyledons of germinating soybean is controlled by the embryonic axis. Plant growth regulators like gibberellic acid, indole acetic acid and 2,4-dichlorophenoxy acetic acid are able to increase the enzyme activity in cotyledons of whole seedlings but not in dissected cotyledons. The control of induction of the enzyme activity during germination by the embryo could be mediated by the elaboration of kinetin.     

Caenorhabditis elegans and Ascaris suum: inhibition of isocitrate lyase by itaconate

The largest forms of isocitrate lyase from Caenorhabditis elegans and Ascaris suum of 543,000 and 549,000 daltons, respectively, can be purified from three- to five-fold in excellent yield by pelleting from extracts at 160,000g for 4 hr. Isocitrate lyase in the pellet is much more stable toward proteolysis. Itaconate which both inhibits isocitrate lyase and suppresses the level of this enzyme in bacteria inhibits the partially purified isocitrate lyase from both C. elegans and A. suum. The inhibition is noncompetitive with respect to d_s-isocitrate at one itaconate concentration. The K_i values at 30 C, pH 7.7, are 19 and 7.3 μM for the enzyme from C. elegans and A. suum, respectively. Itaconate inhibits the growth of C. elegans in random axenic as well as monoxenic cultures. At a concentration of 10 mM, itaconate is more effective in the inhibition of random axenic cultures than is oxalate, maleate, or succinate. At 60 mM itaconate, reproduction of C. elegans larvae is completely abolished.     

Caenorhabditis elegans: Decay of isocitrate lyase during larval development

Changes in the levels of isocitrate lyase, malate synthase, catalase, fumarase, and NADP⁺-isocitrate dehydrogenase have been investigated during larval development of the free-living soil nematode Caenorhabditis elegans in the presence and absence of Escherichia coli. The specific activities of isocitrate lyase, malate synthase, and catalase are maximal at the time of egg hatching and, thereafter, decline during larval development when larvae feed on E. coli, whereas in the absence of E. coli specific activities of the same enzymes increase for 12 hr and subsequently remain constant. There is, however, no change in specific activity of fumarase or NADP⁺-isocitrate dehydrogenase during the same developmental period, in either case. Cycloheximide at 100 μM arrests the decline of isocitrate lyase during development of feeding larvae but has no effect upon the appearance of isocitrate lyase during starvation. The latter is true also for 15 mM itaconate. There is inactivation of isocitrate lyase in crude extracts of frozen worms in comparison to that in analogous extracts prepared from freshly harvested nematodes.     

Bahamaolide A from the marine-derived Streptomyces sp. CNQ343 inhibits isocitrate lyase in Candida albicans

Bahamaolide A, a new macrocyclic lactone isolated from the culture of marine actinomycete Streptomyces sp. CNQ343, was evaluated for its inhibitory activity toward isocitrate lyase (ICL) from Candida albicans. These studies led to the identification of bahamaolide A as a potent ICL inhibitor with IC₅₀ value of 11.82 μM. The growth phenotype of ICL deletion mutants and quantitative RT-PCR analyses indicated that this compound inhibits the ICL mRNA expression in C. albicans under C₂-carbon-utilizing conditions. The present data highlight the potential for bahamaolide A treatment of C. albicans infections via inhibition of ICL activity.     

Estimation of the Mr of isocitrate lyase messenger RNA from Chlorella fusca

The mRNA for the adaptive enzyme isocitrate lyase (ICL) from Chlorella fusca has been identified by fractionation of total poly(A)-containing RNA and in vitro translation followed by immune precipitation. The Mr of ICL mRNA was approximately 8.0 X 10(5), which is in good agreement with a previous estimate obtained by in vivo double-labelling.     

Evidence for the existence of isocitrate lyase activity in methylotrophic Hyphomicrobium strains

Abstract Isocitrate lyase activities were detected in a range of 0.096–0.212 units mg⁻¹ in cell-free extracts of all tested Hyphomicrobium strains grown on methanol as a sole carbon source, although the activities were rapidly lost during storage at 4 °C. When cell-free extracts were incubated with dithiothreitol, after storage the recovery of activity was observed, indicating the involvement of a labile sulfhydryl group in the enzyme. This confirmed the distribution of unstable isocitrate lyase in the genus Hyphomicrobium , and, contrary to previous observations, the operation of the ic ⁺-serine pathway was suggested for the assimilation of one-carbon compounds.     

Purification and characterization of ferredoxin-NADP+ reductase from the green alga Chlorella fusca

Ferredoxin-NADP⁺ reductase (FNR, EC I.18.1.2) from the green algae Chlorella fusca Shihira et Kraus 211–15, was purified to homogeneity. The molecular mass was 36.8 kDa as determined by SDS-polyacrylamide gel electrophoresis. The enzyme exhibits the typical spectrum of a flavoprotein with an absorption maximum at 459 nm and an A_273/459 ratio of 7.2. It contains one mol of FAD per mol of protein and the calculated extinction coefficient is 9.8 m M cm⁻¹. Four different forms of the purified enzyme were detected by isoelectric focusing (pI between 5.4 and 5.9), even when protease inhibitors were used during the first steps of the purification. Kinetic parameters were determined for several FNR-catalyzed reactions. NADP⁺ photoreduction gave comparable rates when either ferredoxin or flavodoxin was used.     

The cytoplasmic pH in photosynthesizing cells of the green alga Chlorella fusca,measured by P-31 NMR spectroscopy

P-31 NMR investigations were performed with the green alga Chlorella fusca under anaerobic conditions in the dark and in the light.In spectra of cells in the dark the signal of intracellular, nonvacuolar P_i indicates a pH in its chemical environment of 7.0–7.2. Upon illumination this signal looses intensity and shifts to lower field, corresponding to a pH of 7.7. Further downfield no other signal that could be attributed to a P_i-pool in more alkaline environment was detected. By the use of 2-deoxyglucose-6-phosphate as an indicator of cytoplasmic pH, this P_i-signal was assigned to the cytoplasm. The pH increase in the cytoplasm upon transfer of cells from the dark to the light is the same as that previously observed upon transfer of cells from anaerobic to aerobic conditions.In cells performing only cyclic photophosphorylation the cytoplasmic pH is lower than in photosynthesizing cells but still 0.2 pH units higher than in the cells in the dark. The reasons for the missing of a signal of stromal P_i and for the difference in cytoplasmic pH in photosynthesizing cells and those capable only of cyclic photophosphorylation are discussed.Non-standard abbreviations 2dG 2-Deoxyglucose - dG-6-P 2-deoxyglucose-6-phosphate - DCMU 3,4-dichlorophenyl-dimethylurea - MOPSO 3-(N-morpholino)-2-hydroxypropane sulfonic acid - P-31 NMR P-31 nuclear magnetic resonance     

Isolation and characterization of thiosulfate reductases from the green alga Chlorella fusca

Thiosulfate-reductase activity (TSR) measured as sulfide release from thiosulfate was detected in crude extracts of Chlorella using dithioerythritol (DTE) as electron donor. Purification of this activity by ammonium-sulfate precipitation between 35% and 80% followed by Sephadex G-50 gel filtration, diethylaminoethyl-cellulose chromatography, and gel filtration on Biogel A 1.5 M led to four distinct proteins having molecular weights of: TSR I, 28000; TSR II, 26500; TSR III_a, 55000; TSR III_b, 24000 daltons. These thiosulfate reductases were most active with DTE; the monothiols glutathione, l-cysteine, and -mercaptoethanol had little activity towards this system. The following pH optima were obtained: for TSR I and TSR II, 9.0; for TSR III_a, 8.5; and for TSR III_b, 9.5. The apparent-K_m data for DTE and thiosulfate were determined to: TSR I, 0.164 mmol·l^-1 and TSR II, 0.156 mmol·l^-1; K_mDTE TSR I, 1.54 mmol·l^-1 and TSR II 1.54 mmol·l^-1. The thiosulfate reductases III_a and III_b were further stimulated by addition of thioredoxin. All TSR fractions catalyzed SCN formation from thiosulfate and cyanate and thus had rhodanese activity; this activity, however, could only be detected in the presence of thiols.Abbreviations DTE dithioerythritol - TSR thiosulfate reductase Dedicated to Professor Dr. Hubert Ziegler on the occasion of his 60th birthday     

Control of metabolic interconversion of isocitrate dehydrogenase between the catalytically active and inactive forms in Escherichia coli

The enzymic interconversion of Escherichia coli isocitrate dehydrogenase (ICDH) between the catalytically active and inactive forms is mediated through the activities of ICDH-kinase/phosphatase in response to changes in the metabolic environment. In this study, the use of mutant strains devoid of isocitrate lyase ( aceA:: Tn10 ) and pyruvate dehydrogenase activities revealed that the signal which triggers the reversible inactivation of ICDH in vivo is not directly related to acetate itself, but rather to the need to maintain high intracellular levels of isocitrate and free co-enzyme A. The use of these mutants also revealed, rather unexpectedly, that acetate grown cells contain more ICDH protein than those grown with other carbon sources and that the catalytic activity of ICDH kinase/phosphatase is in excess of cellular demands. Furthermore, this study also revealed the presence of a 50-kDa (±2 kDa) acetate-specific polypeptide, the identity of which has yet to be established.     

Microbody transition in greening watermelon cotyledons Double immunocytochemical labeling of isocitrate lyase and hydroxypyruvate reductase

Microbody transition during the greening of watermelon cotyledons (Citrullus vulgaris Schrad.) was studied by double immunocytochemical labeling of the glyoxysomal marker enzyme isocitrate lyase and the peroxisomal marker enzyme hydroxypyruvate reductase. In order to analyze the immunocytochemistry, developmental stages representing the glyoxysomal, microbodytransition and peroxisomal stages were chosen, taking into account the time course of enzyme activity and the amounts of the respective antigens. It was shown that during microbody transition, between 83 and 91% of all the tested microbodies contained isocitrate lyase as well as hydroxypyruvate reductase, which was significantly higher than in the glyoxysomal and peroxisomal stages of development. Comprehensive controls precluded labeling artifacts. Our results support the one-population hypothesis first proposed by Trelease et al. (1971, Plant Physiol. 48, 461–465).Abbreviations ICJ isocitrate lyase - HPR hydroxypyruvate reductase - pAg small protein A-gold complex - pAG large protein A-gold complex     

Uptake and metabolism of taurine in the green alga Chlorella fusca

Taurine entered the alga Chlorella fusca Shihira et Krauss strain 21l-8b via a pH and energy-dependent system ("permease"). Transport followed triphasic kinetics from 10⁻⁶ to 10⁻² M with K_m values for taurine of 5.4 × 10⁻⁵, 4.1 × l0⁻⁴ and l.5 × 10⁻³ M. This uptake system was specific for sulfonic acids and showed no affinity for α- and β -amino acids or Na⁺; thus the permease of C. fusca is different from all known taurine transport systems with respect to structural specificity and lack of Na⁺ -dependence. Uptake was not observed in sulfate-grown algae but developed as a response to sulfate limitation within 2 h. Sulfate addition caused a rapid decline in taurine transport capacity. Labeled taurine was rapidly metabolized in C. fusca to sulfate and ethanolamine, suggesting oxidative hydrolysis as the mechanism of C-S bond cleavage. Further incorporation of these catabolic products in C - and S -metabolism was demonstrated. Taurine catabolism was also detected in other green algae and some cyanobacteria.     

The purification and properties of isocitrate lyase from Chlorella

1. Isocitrate lyase (threo-d(s)-isocitrate glyoxylate-lyase, EC 4.1.3.1) has been purified from acetate-adapted cells of Chlorella pyrenoidosa. 2. The final preparation was homogeneous by the criteria of sedimentation, diffusion and polyacrylamide-gel electrophoresis. 3. The sedimentation coefficient (S(20,w)) was 9.04x10(-13)sec. and the diffusion coefficient (D(20,w)) 4.62x10(-7)cm.(2)/sec.; from these values the molecular weight of the enzyme was calculated to be 170000 and its Stokes radius to be 4.63x10(-7)cm. 4. The elution of the enzyme from Sephadex G-100 was studied and estimates of molecular weight and Stokes radius were obtained from the elution data. 5. The turnover number of the enzyme was 5950moles of glyoxylate formed/min./mole of enzyme at 30 degrees . 6. With threo-d(s)(+)-isocitrate as substrate, the K(m) of the enzyme was 0.023mm.     

Glucose-induced inactivation of isocitrate lyase in Aspergillus nidulans

     The existence of a second mechanism of catabolite control of isocitrate lyase of Aspergillus nidulans, in addition to the carbon catabolite repression phenomenon recently reported was analysed. Isocitrate lyase was rapidly and specifically inactivated by glucose. The inactivation was irreversible at all stages in the presence of cycloheximide, showing that reactivation depends on de novo protein synthesis. In addition, analysis of glucose-induced inactivation of isocitrate lyase in a creA ^d -30 strain showed that the creA gene is not involved in this process. Received: 13 May 1994 / Accepted 12 August 1994