RHYTHMIC REGULATION OF THE LIGHT-HARVESTING CHLOROPHYLL alb PROTEIN and THE SMALL SUBUNIT OF RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE mRNA IN RYE SEEDLINGS

In etiolated rye seedlings transferred to light the expression of chlorophyll a/b binding protein mRNA varies when the seedlings are grown in a day/night cycle. The fluctuation pattern follows a circadian rhythm. Exposure of 4-day old etiolated seedlings to continuous white light revealed two maxima within the first 24 h before the 24 h cycle period appeared. These first two maxima are also observable after a pulse of white light or after a pulse of red light. These results indicate a possible involvement of phytochrome in the endogenous regulation of the rhythm.     

THE PHOTOMORPHOGENETIC CONTROL OF THE DEVELOPMENT OF PLASTID GLYCERALDEHYDE-PHOSPHATE DEHYDROGENASE AND PHOSPHORIBULOKINASE ACTIVITIES IN COTYLEDONS OF MUSTARD SEEDLINGS

The plastid glyceraldehyde-phosphate dehydrogenase and phosphoribulokinase of mustard cotyledon extracts were activated by preincubation with ATP and with ATP and dithiothreitol respectively. By in vitro activation prior to assay, it was possible to determine the potential activities, which appear to have been directly proportional to the amount of each enzyme protein present. In this way it was possible to deduce the net synthesis of these two enzymes. The induction of synthesis of glyceraldehyde-phosphate dehydrogenase and phosphoribulokinase, by continuous far red or white light were similar hut net synthesis in continuous far red continued longer for glyceraldehyde-phosphate dehydrogenase than for phosphoribulokinase. The kinetics of the development of the glyceraldehyde-phosphate dehydrogenase potential activity were very close to those reported by Bruning et al. (1975) for this enzyme. The data do not permit elimination of either the single switch or multiple switches hypotheses for the action of phytochrome. Continuous white illumination gave results similar to those for continous far red for the net synthesis of the two enzymes but it was more effective than far-red in bringing about enzyme activation in vivo.     

THE CONTROL OF METABOLISM BY BLUE LIGHT IN Acetabularia mediterranea—II. SELECTIVE TRANSLATIONAL CONTROL AND DIFFERENTIAL DEGRADATION OF ENZYMES

Abstract— During prolonged continuous irradiation with red light the specific activity of uridine 5'-diphosphoglucose (UDPG) pyrophosphorylase (uridine 5'-triphosphate: glucose 1-phosphate uridylyl-transferase EC 2.7.7.9) decreased in Acetabularia mediterranea Lamouroux (=A. acetabulum (L.) Silva). Subsequent blue light restored the original activity within a comparatively short period of 3 to 4 days. Computer-aided quantitative evaluation of density labelling experiments showed that the synthesis of the enzyme was accelerated about four-fold during the period of activation by blue light. A similar increase in the rate of synthesis was found for hydroxypyruvate reductase (EC 1.1.1.81), a control enzyme that showed no blue light-dependent changes in the specific activity under these conditions. The increase in the rate of enzyme synthesis was caused by an overall stimulation of the cytosolic translation. Degradation of UDPG pyrophosphorylase was unaffected by blue light, while the half life of hydroxypyruvate reductase was shortened about two-fold compared to continuous red light. Thus, degradation of proteins appears to be selectively light dependent in Acetabularia.
Model calculations for enzyme amount and enzyme synthesis were carried out using the measurements of enzyme activity, rates of cytosolic protein synthesis, and degradation constants of the enzymes. Assuming that activities represented amounts of the given enzymes, these calculations indicated a selective activation of UDPG pyrophosphorylase synthesis by blue light since it did not coincide with the overall stimulation of protein synthesis in the cytosol, in contrast to hydroxypyruvate reductase.     

Transketolase from human erythrocytes. Purification and properties

Human erythrocyte transketolase (sedoheptulose-7-phosphate: D-glyceraldehyde-3-phosphate glycolaldehyde-transferase) was purified 8200-fold by adsorption onto hydroxylapatite, DEAE-cellulose treatment, acetone fractionation, and chromatography on Sephadex G-100. The purified transketolase could not be separated from glyceraldehyde-3-phosphate dehydrogenase, whereas the latter enzyme could be isolated in a pure state. Its homogeneity is suggested by sedimentation velocity, sedimentation equilibrium, and acrylamide electrophoresis. A molecular weight of 136000 was found. The physicochemical properties of glyceraldehyde-3-phosphate dehydrogenase and transketolase are very similar. A molecular weight of 136000 is suggested for transketolase, although gel filtration with Sephadex G-100 gave only 104000 ± 10%. This discrepancy is a reflection of an interaction of transketolase with the gel filtration medium. The isoelectric point for transketolase as well as for glyceraldehyde-3-phosphate dehydrogenase, as determined by isoelectric focussing, was found to be around 8.5. The activity of the enzyme is close to the maximum for pH 7.5 to pH 8.6. Additions of thiamine pyrophosphate or other cofactors do not influence the activity. Several divalent cations were tested. Sulfate and phosphate inhibit transketolase approximately to 50% between 50 and 100 mM concentration. Thiamine was present in transketolase, as shown by a microbiological assay and by the thiochrome reaction. The activation energy for the formation of sedoheptulose-7-phosphate from xylulose-5-phosphate was estimated from rate measurements to be 11.2 kcal/mole in the temperature range from 5° to 55°.     

EFFECTS OF PHASIC AND TONIC LIGHT INPUTS ON THE ORCADIAN ORGANIZATION OF THE SQUIRREL MONKEY

Abstract— The organization of the circadian timing system in Saimiri sciureus was probed using the phasic (abrupt transition) and tonic (continuous action) effects of light intensity. The behavior of the simultaneously monitored circadian rhythms of feeding behavior, colonic temperature, and urinary potassium excretion was studied in response to the phasic effects of (a) an abrupt 8-h phase delay in the light–dark (LD) cycle and (b) a series of non-24 h LD cycles ( T = 18 to 30 h). These studies demonstrated that the feeding and temperature rhythms were more tightly coupled to the light-dark cycle than was the rhythm of urinary potassium excretion. The tonic effects of constant levels of illumination confirmed this conclusion. In constant light, internal desynchronization spontaneously occurred in 25% of animals with the potassium rhythm exhibiting a period quite different from that of the feeding and colonic temperature rhythms. Thus, the response of the internal circadian timekeeping system to phasic and tonic light inputs shows that the system in this species comprises at least two potentially independent oscillators with differential light sensitivities.     

CONTROL OF PHOTOSYNTHETIC REDUCTANT: THE ROLE OF LIGHT AND TEMPERATURE ON SUSTAINED HYDROGEN PHOTOEVOLUTION BY Chlamydomonas sp. IN AN ANOXIC,CARBON DIOXIDE-CONTAINING ATMOSPHERE*

–Sustained hydrogen photoevolution from Chlamy domonas reinhardtii and C. Moewusii was measured under an anoxic, CO₂-containing atmosphere. It has been discovered that light intensity and temperature influence the partitioning of reductant between the hydrogen photoevolution pathway and the Calvin cycle. Under low incident light intensity (1-3 W m^-2) or low temperature (approx. 0°C), the flow of photosynthetic reductant to the Calvin cycle was reduced, and reductant was partitioned to the hydrogen pathway as evidenced by sustained H₂ photoevolution. Under saturating light (25 W m^-2) and moderate temperature (20±5°C), the Calvin cycle became the absolute sink for reductant with the exception of a burst of H₂ occurring at light on. This burst of H₂ corresponded to the expression of about 450 electrons for each photosynthetic electron transport chain. These results suggest that the hydrogen pathway and the Calvin cycle compete for reductant under anoxic conditions and that partioning between the two pathways can, to a certain extent, be controlled by the appropriate choice of experimental conditions.     

Selective inhibition of Trypanosoma cruzi GAPDH by "bi-substrate" analogues

A new series of "bi-substrate" analogues have been synthesized as potential inhibitors of the glyceraldehyde-3-phosphate dehydrogenase and one lead compound has been identified that inhibits the enzyme from Trypanosoma cruzi with good affinity and very high (50-fold) specificity.     

Development and characterization of an immobilized enzyme reactor based on glyceraldehyde-3-phosphate dehydrogenase for on-line enzymatic studies

The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been extensively studied as a target for new drugs to be used in the treatment of various parasitic diseases. The standard approach to the determination of GAPDH activity utilizes solubilized free enzyme and is limited by the enzyme's low stability. In the current study the stability of GAPDH was significantly increased through the covalent immobilization of the enzyme on a wide-pore silica support containing glutaraldehyde (Glut-P). The optimal conditions for the immobilization were: 100 mg Glut-P stationary phase, approximately 150 microg of enzyme dissolved in pyrophosphate buffer (15 mM, pH 8.5). The mixture was gently agitated for 6 h at 4 degrees C. Under these conditions 91.3% of protein was immobilized on 100 mg of Glut-P support with retention of 2.97% of the initial enzymatic activity. The activity of the immobilized GAPDH was stable for over 30 days. The GAPDH-Glut-P stationary phase was packed into a glass column to produce a GAPDH immobilized enzyme reactor (GAPDH-IMER). The activity and kinetic parameters of the GAPDH-IMER were investigated and the results demonstrated that the enzyme retained its activity and sensitivity to the competitive inhibitor agaric acid.     

Effects of Oxygen Limitation on Xylose Fermentation, Intracellular Metabolites, and Key Enzymes of Neurospora crassa AS3.1602

The effects of oxygen limitation on xylose fermentation of Neurospora crassa AS3.1602 were studied using batch cultures. The maximum yield of ethanol was 0.34 g/g at oxygen transfer rate (OTR) of 8.4 mmol/L·h. The maximum yield of xylitol was 0.33 g/g at OTR of 5.1 mmol/L·h. Oxygen limitation greatly affected mycelia growth and xylitol and ethanol productions. The specific growth rate (μ) decreased 82% from 0.045 to 0.008 h⁻¹ when OTR changed from 12.6 to 8.4 mmol/L·h. Intracellular metabolites of the pentose phosphate pathway, glycolysis, and tricarboxylic acid cycle were determined at various OTRs. Concentrations of most intracellular metabolites decreased with the increase in oxygen limitation. Intracellular enzyme activities of xylose reductase, xylitol dehydrogenase, and xylulokinase, the first three enzymes in xylose metabolic pathway, decreased with the increase in oxygen limitation, resulting in the decreased xylose uptake rate. Under all tested conditions, transaldolase and transketolase activities always maintained at low levels, indicating a great control on xylose metabolism. The enzyme of glucose-6-phosphate dehydrogenase played a major role in NADPH regeneration, and its activity decreased remarkably with the increase in oxygen limitation.     

Bilirubin-sensitized photoinactivation of enzymes in the isolated membrane of the human erythrocyte.

Abstract— Bilirubin has been found to sensitize the photodynamic inactivation of several enzymes in the isolated membrane (ghost) of the human red cell. When ghosts (pH 8.0, 10°C) + bilirubin (0.1 mM) were irradiated with blue light (350 Wm^-2), the activity of glyceraldehyde 3-phosphate dehydrogenase decayed with t_1/2? 15 min. No effect was observed in the absence of pigment or with incident yellow light. Diazabicyclo-octane (DABCO) sharply reduced the inactivation rate, suggesting that ¹O₂ is involved. Sodium dodecyl sulfate-gel electrophoresis of ghosts containing fully inactivated glyceraldehyde 3-phosphate dehydrogenase revealed no change in the polypeptide band corresponding to the subunit of the enzyme. Solubilized enzyme, which was similarly photosensitive, could be partially protected by nicotinamide adenine dinucleotide or glyceraldehyde 3-phosphate. The integral enzymes Mg²⁺-ATPase, Na⁺, K⁺-ATPase, and acetylcholinesterase were also affected. Under the above conditions and bilirubin = 0.37 mM, these enzymes were photoinactivated in first-order fashion, k? 2, 1.2 and 0.2 h^-1, respectively. The rate of decay of total ATPase was found to vary as the square root of the bilirubin concentration over the range 7–370 μM. At a fixed bilirubin concentration (0.37 mM), this rate was also shown to be directly proportional to light intensity. Inasmuch as the —SH content of bilirubin-containing ghosts diminished during irradiation, oxidation of essential cysteine residues could be responsible for the inactivation of some of the enzymes studied.     

Photodynamically induced effects in colon carcinoma cells (WiDr) by endogenous photosensitizers generated by incubation with 5-aminolaevulinic acid.

Human adenocarcinoma cells of the line WiDr have been treated with 2 mM 5-aminolaevulinic acid (5-ALA) in the presence of 10% foetal calf serum. The treatment induces a linear accumulation of protoporphyrin IX (PpIX) for at least 7.5 h. After 7.5 h of incubation about 45% of the PpIX accumulated is cell-bound, while the rest is found in the medium (25%) or lost from the cells during washing with phosphate-buffered saline (30%). Exposure to white light at an intensity of 30 W/m2 for 18 min results in 95% reduction of clonogenicity in cells treated with 2 mM 5-ALA for 3.5 h. The enzymatic activities of enzymes located in cytosol (glyceraldehyde 3-phosphate dehydrogenase and lactate dehydrogenase) and lysosomes (acid phosphatase and beta-glucuronidase) are not influenced by a 5-ALA and light treatment inactivating about 35% of the cells. The MTT assay, which reflects mitochondrial dehydrogenase activity, but not succinate dehydrogenase, is partly inhibited by the same treatment. Treatment with 5-ALA in the absence of light increases O2 consumption by a factor of two, while the O2 consumption is inhibited when 5-ALA treatment is combined with exposure to light. In addition, 5-ALA and light exposure enhance accumulation of rhodamine 123 by 40% and reduce the intracellular ATP level by 25%. Confocal laser scanning microscopical analysis indicates granular perinuclear localization of the PpIX formed by 5-ALA treatment. In conclusion, photodynamic treatment using 5-ALA as a prodrug induces damage to mitochondrial function without inhibiting lysosomal and cytosolic marker enzymes.     

Development and characterization of an immobilized enzyme reactor (IMER) based on human glyceraldehyde-3-phosphate dehydrogenase for on-line enzymatic studies

Immobilized enzyme reactors (IMERs) for on-line enzymatic studies are useful tool to select specific inhibitors and may be used for direct determination of drug-receptor binding interactions and for the rapid on-line screening to identify specific inhibitors. This technique has been shown to increase the stability of enzymes. The enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays an important role in the life cycle of the Trypanosoma cruzi and it has become a key target in the drug discovery program for Chagas' disease. Crystallographic studies have indicated that there are significant inter-species differences in GAPDH activity and sensitivity. For example the active sites of GAPDH in T. cruzi and humans differ by a substitution of ASP(210) (T. cruzi) by Leu(194) in human. Based on this information we initiated the study to develop optimal conditions for the covalent immobilization of the human GAPDH enzyme on a modified capillary support (400 mm x 0.10 mm). The chromatographic separation of NAD from NADH was achieved using a RP-Spherex-diol-OH (10 cm x 0.46 cm, 10 microm, 100 A) column. By using multidimensional HPLC chromatography system it was possible to investigate the activity and kinetic parameters of the GAPDH-IMER. The values obtained for D-GA3P and NAD were K(m)=3.5+/-0.2 mM and 0.75+/-0.04 mM, respectively, and were compared with values obtained with the free enzyme. The activity of the immobilized GAPDH has been preserved for over 120 days.     

Interaction of polyanions with basic proteins, 2(a) : influence of complexing polyanions on the thermo-aggregation of oligomeric enzymes

The ability of synthetic polyanions to suppress thermo-aggregation of the oligomeric enzymes (glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, and aspartate aminotransferase) has been established. The ability of the polyanions to reduce the thermo-aggregation increased in the order poly(methacrylic acid) < poly(acrylic acid) < sodium poly(styrene sulphonate), which agreed well with the increase, in the same order, of the charge density of the chains. The lengthening of the chains, as well as the rise in their relative content, resulted in an increase of the ability to reduce thermo-aggregation, mentioned above. Complete prevention of the enzyme aggregation was achieved when highly charged polyanions of a relatively high degree of polymerization were used in a concentration sufficient to solubilize the protein. Complexing with the polyanions prevented thermo-aggregation of the enzymes, but not their thermo-denaturation. The adverse effect of the complexing polyanions on the catalytic activity was reduced by the addition of a synthetic polycation, which resulted in a significant reactivation (up to 40%) of the enzyme. The possibility of preventing the thermo-aggregation of enzyme molecules and then partly restoring the enzyme activity, appears to be of particular interest when studying the aggregation mechanism of proteins that are prone to form the amyloid structures responsible for the development of neurodegenerative diseases like Alzheimer's disease, bovine spongiform encephalopathy and Huntington disease. This finding can also be considered as an important step in the creation of artificial chaperones.     

Partial purification of glucose 6-phosphate dehydrogenase and phosphofructokinase from rat erythrocyte haemolysate by partitioning in aqueous two-phase systems

Glucose 6-phosphate dehydrogenase shows a high partition coefficient in poly-(ethylene glycol)-dextran aqueous two-phase systems in comparison with those for 6-phosphogluconate dehydrogenase, phosphofructokinase and the bulk of proteins present in rat erythrocyte haemolysates. As a consequence, fractions highly enriched in glucose 6-phosphate dehydrogenase can be obtained after multiple partitions in the above systems with a counter-current distribution procedure. Phosphofructokinase shows a high affinity for Cibacron Blue and, as a result, the enzyme can be extracted in the top phase of poly(ethylene glycol)-dextran systems containing Cibacron Blue-poly(ethylene glycol) (affinity systems). The efficiency for the purification of the enzymes by partitioning is increased up to 10-fold when enzyme-rich fractions, obtained by precipitation with poly(ethylene glycol), are used instead of original haemolysate. The recovery of enzyme activities is near 100% in both instances.     

2-DE and MS analysis of interactions between Lactobacillus fermentum I5007 and intestinal epithelial cells

Lactobacillus is a probiotic commonly used for supplementation to human and animal diets. In this study, we used 2-DE and MS to analyze changes in the proteomes of Lactobacillus and intestinal epithelial cells in two model systems. The in vivo and in vitro models were involved the inoculation of Lactobacillus fermentum I5007 into the rabbit jejunum for 4 h and the culture of the bacterium with Caco-2 cells for 1 h, respectively. Our results indicate that, after exposure to the intestinal environment, the bacterium exhibited decreases in key enzymes involved in energy metabolism (e.g., lactate dehydrogenase, dihydrolipoamide dehydrogenase, and nicotinate phosphoribosyltransferase) and amino acid metabolism (e.g., arginyl-tRNA synthetase and aspartate-semialdehyde dehydrogenase), but increases in glycoside hydrolase (an enzyme for mucin degradation) and fructose-6-phosphate phosphoketolase (an enzyme of the pentose phosphate pathway). In response to an interaction with L. fermentum I5007, Caco-2 cells showed changes in proteins that were beneficial for gut integrity, including voltage-dependent anion channel 1, glutathione transferase, and heat shock protein gp96. On the basis of their functions, we suggest that these proteins serve as useful biomarkers for metabolic changes in Lactobacillus and intestinal epithelial cells in response to their interactions.     

PHOTOCHEMICAL REACTIVITY OF MOLECULES TRAPPED IN SOL-GEL GLASSES*

The effects of different wavelengths of light on period, phase shifting, entrainment and after-effects of the circadian clock of the motile marine dinoflagellate Gonyaulax polyedra are described. Phase shifting and entrainment by light can be explained by the action of a single blue sensitive light input pathway. However, tonic effects of light on the period under free-running conditions, and also after-effects on period resulting from single 4 h light exposures, appear to involve two input pathways with different absorption and temperature characteristics. These results suggest different mechanisms for the control of phase and period of the circadian clock.     

Principles of multienzyme purifications by affinity chromatography

Recently in our laboratory, up to 20 different enzymes and their genetic variants have been purified from mouse andDrosophila by affinity chromatography. By virtue of the specific coenzyme requirements, up to ten different enzymes could be copurified from a single tissue extract either by biospecific elutions with different coenzymes or inhibitors, or by sequential passages of the extract through several cofactor-related affinity columns. Important principles were developed to purify enzymes exhibiting low affinity to the affinity columns. By “affinity filtration” of the extract through the affinity column, enzymes of low affinity can be retarded and separated effectively from strongly bound and nonadsorbed proteins. By the “saturation readsorption” procedure, enzymes of low affinity could be effectively separated from those of high affinity by overloading of the extracts on the affinity columns. Readsorption of the leaked low affinity enzymes to a second affinity column often results in better enzyme purification because of the elimination of competitive high affinity enzymes. With the application of these principles, the following enzymes and their genetic variants were highly purified via a single- or two-step affinity column procedure: lactate dehydrogenase-A, lactate dehydrogenase-B, lactate dehydrogenase-X, phosphoglycerate kinase-A, phosphoglycerate kinase-B, cytoplasmic and mitochondrial isocitrate dehydrogenase, malate dehydrogenase, malic enzyme, glucose-6-phosphate dehydrogenase, glutathione reductase, phosphoglucose isomerase and pyruvate kinase from mouse tissues; alcohol dehydrogenase, malate dehydrogenase, α-glycerol-phosphate dehydrogenase, malic enzyme, and glucose-6-phosphate dehydrogenase fromDrosophila.     

PHOTO ACTIVATION OF ISOFLAVONOID PHYTOALEXINS: INVOLVEMENT OF FREE RADICALS

Abstract— Upon irradiation with ultraviolet light the isoflavonoid phytoalexins phaseollin, 3,6a, 9-trihydroxypterocarpan, glyceollin, tuberosin and pisatin, but not medicarpin, brought about inactivation ofglucose–6-phosphate dehydrogenase in an in vitro assay system. Photoinactivation of the enzyme by photoactivated pisatin in air-saturated solutions was hardly affected by singlet oxygen quenchers such as NaN₃, bovine serum albumin, histidine or methionine. Neither addition of the hydroxyl radical scavengers mannitol, Na-benzoate and ethanol nor the presence of catalase or supcroxide dismutase protected the enzyme against photoinactivation, suggesting that OH, H₂O₂ and O₂ are not the reactive oxygen species involved. However, the free radical scavenger S-(2-amino-ethyl)isothiouronium bromide hydrobromide (AET) protected the enzyme against inactivation by photoactivated pisatin. Direct evidence for the generation of free radicals was obtained by ESR measurements of solutions of phaseollin, pisatin and medicarpin in hexane irradiated with ultraviolet light in the presence or absence of O₂. Phaseollin produced the most stable free radicals, whereas medicarpin hardly gave rise to free radical formation; pisatin took a somewhat intermediate position by producing a strong ESR signal which, however, decayed rather quickly. Photodegradation of all phytoalexins, except for medicarpin, was accompanied with loss of fungitoxicity, as shown in thin-layer chromatographic bioassays, and formation of new products.
These results indicate free radical formation as the causative process for photoinactivation of enzymes by photoactivated isoflavonoid phytoalexins.     

Efficiency of superoxide anions in the inactivation of selected dehydrogenases

The most ubiquitous of the primary reactive oxygen species, formed in all aerobes, is the superoxide free radical. It is believed that the superoxide anion radical shows low reactivity and in oxidative stress it is regarded mainly as an initiator of more reactive species such as OH and ONOO^‐.In this paper, the effectiveness of inactivation of selected enzymes by radiation-generated superoxide radicals in comparison with the effectiveness of the other products of water radiolysis is examined. We investigate three enzymes: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), alcohol dehydrogenase (ADH) and lactate dehydrogenase (LDH).We show that the direct contribution of the superoxide anion radical to GAPDH and ADH inactivation is significant. The effectiveness of the superoxide anion in the inactivation of GAPDH and ADG was only 2.4 and 2.8 times smaller, respectively, in comparison with hydroxyl radical. LDH was practically not inactivated by the superoxide anion.Despite the fact that the studied dehydrogenases belong to the same class of enzymes (oxidoreductases), all have a similar molecular weight and are tetramers, their susceptibility to free-radical damage varies. The differences in the radiosensitivity of the enzymes are not determined by the basic structural parameters analyzed. A significant role in inactivation susceptibility is played by the type of amino acid residues and their localization within enzyme molecules.