Purification and properties of glycolate oxidase from plants with different photosynthetic pathways: Distinctness of C4 enzyme from that of a C3 species and a C3–C4 intermediate

Glycolate oxidase (GO; EC 1.1.3.1) was purified from the leaves of three plant species:Amaranthus hypochondriacus L.(NAD-ME type C₄ dicot),Pisum sativum L. (C₃ species) andParthenium hysterophorus L. (C₃–C₄. intermediate). A flavin moiety was present in the enzyme from all the three species. The enzyme from the C₄ plant had a low specific activity, exhibited lower K_M for glycolate, and required a lower pH for maximal activity, compared to the C₃ enzyme. The enzyme from the C₄ species oxidized glyoxylate at <10% of the rate with glycolate, while the GO from the C₃ plant oxidized glyoxylate at a rate of about 35 to 40% of that with glycolate. The sensitivity of GO from C₄ plant to -hydroxypyridinemethane sulfonate, 2-hydroxy-3-butynoate and other inhibitors was less than that of the enzyme from C₃ source. The properties of GO fromParthenium hysterophorus, were similar to those of the enzyme fromPisum sativum. The characteristics of glycolate oxidase from leaves of a C₄ plant,Amaranthus hypochondriacus are different from those of the C₃ species or the C₃–C₄ intermediate.     

Light Modulation and Localization of Sucrose Phosphate Synthase Activity between Mesophyll Cells and Bundle Sheath Cells in C(4) Species

Experiments were conducted with several Panicum species (representing the different C₄ subtypes) to examine the light modulation of sucrose phosphate synthase (SPS) activity and the effect of illumination on the distribution of SPS activity between mesophyll cells (MC) and bundle sheath cells (BSC). Activity of SPS in the light decreased in the order: C₄ > C₃-C₄ intermediate > C₃. In illuminated leaves, SPS activities were similar among the three C₄ subtypes, but SPS activity was higher for NAD-malic enzyme (NAD-ME) species with centripetal chloroplasts in BSC (NAD-ME(P) species) than for NAD-ME species with centrifugal chloroplasts in BSC (NAD-ME(F) species). Transfer of plants into darkness for 30 minutes resulted in decreased SPS activity for all species tested except Panicum bisulcatum (C₃ species) and Panicum virgatum (NAD-ME(P) species) which showed little or no change. All C₄ subtypes had some SPS activity both in MC and BSC. In the light, SPS activity was mainly in the MC for NADP-ME, NAD-ME(F) and phosphoenolpyruvate carboxykinase species, while it was mainly in the BSC for NAD-ME(P) species. In the dark, for all C₄ subtypes, SPS activity in the MC was decreased to a greater extent than that in the BSC. It is intriguing that NAD-ME(F) and NAD-ME(P) species differed in the activity and distribution of SPS activity between MC and BSC, although they are otherwise identical in the photosynthetic carbon assimilation pathway. Diurnal changes in SPS activity in the MC and BSC were also examined in maize leaves. SPS activity in the MC in maize leaves was high and relatively constant throughout the middle of the light period, dropped rapidly after sunset and increased again prior to the light period. On the other hand, SPS activity in the BSC was lower and changed more coincidently with light intensity than that in the MC. The results suggested that light activation of SPS activity located in the BSC may require higher irradiance for saturation than the SPS in the MC. We conclude that SPS may function in both MC and BSC for sucrose synthesis in the light, particularly at high light intensity, while in the dark, the major function may be in the BSC during starch degradation.     

Physical,chemical, and regulatory properties of glycolate oxidase in C<Subscript>3</Subscript> and C<Subscript>4</Subscript> plants

Physical, chemical, and regulatory properties of glycolate oxidase (GO) isolated from the leaves of C₄ and C₃ plants (Zea mays L., cv. Voronezhskaya 76 and Glycine max (L.) Merr., cv. Pripyat’, respectively) were studied. The homogenous preparations were obtained by multistage enzyme purification from soybean leaves and maize mesophyll and bundle sheath. The glycolate oxidase (GO) preparations obtained consisted of two types of subunits, 37 and 44 kD. The GO isolated from C₃ plant leaves had many in common with that extracted from C₄ plant bundle sheath as regards physical, chemical, and catalytic properties. The primary function of GO in both plant types is metabolism of glycolate, which is a product of ribulosebisphosphate oxalacetic acid oxidation and is used by plants for biosynthesis of hydrocarbons and amino acids.     

Glutamine synthetase and glutamate dehydrogenase isoforms in maize leaves: localization, relative proportion and their role in ammonium assimilation or nitrogen transport

 Mesophyll cells (MCs) and bundle-sheath cells (BSCs) of leaves of the C₄ plant maize (Zea mays L.) were separated by cellulase digestion to determine the relative proportion of the glutamine synthetase (GS; EC 6.3.1.2) or the NADH-glutamate dehydrogenase (GDH; EC 1.4.1.2) isoforms in each cell type. The degree of cross-contamination between our MC and BSC preparations was checked by the analysis of marker proteins in each fraction. Nitrate reductase (EC 1.6.6.1) proteins (110 kDa) were found only in the MC fraction. In contrast, ferredoxin-dependent glutamate synthase (Fd-GOGAT; EC 1.4.7.1) proteins (160 kDa) were almost exclusively present in the BSC fraction. These results are consistent with the known intercellular distribution of nitrate reductase and Fd-GOGAT proteins in maize leaves and show that the cross-contamination between our MC and BSC fractions was very low. Proteins corresponding to cytosolic GS (GS-1) or plastidic GS (GS-2) were found in both the MC and BSC fractions. While equal levels of GS-1 (40 kDa) and GS-2 (44 kDa) polypeptides were present in the BSC fraction, the GS-1 protein level in the MC fraction was 1.8-fold higher than the GS-2 protein pool. Following separation of the GS isoforms by anion-exchange chromatography of MC or BSC soluble protein extracts, the relative GS-1 activity in the MC fraction was found to be higher than the relative GS-2 activity. In the BSC fraction, the relative GS-1 activity was very similar to the relative GS-2 activity. Two isoforms of GDH with apparent molecular weights of 41 kDa and 42 kDa, respectively, were detected in the BSC fraction of maize leaves. Both GDH isoenzymes appear to be absent from the MC fraction. In the BSCs, the level of the 42-kDa GDH isoform was 1.7-fold higher than the level of the 41-kDa GDH isoform. A possible role for GS-1 and GDH co-acting in the synthesis of glutamine for the transport of nitrogen is discussed. Received: 25 January 2000 / Accepted: 30 March 2000     

Glutamine synthetase from mesophyll and bundle sheath maize cells: isoenzyme complements and different sensitivities to phosphinothricin

 Anion-exchange FPLC has been used to resolve the isoforms of glutamine synthetase (GS, EC 6.3.1.2) from Zea mays mesophyll (MC) and bundle sheath cells (BSC). Two different isoforms were detected in both types of photosynthetic cells. The predominantly active isoform was GS1 (61%) in MC and GS2 (67%) in BSC. The relative contribution of GS1 and GS2 to the overall GS activity in BSC in maize here reported resembles the proportion described for most C3 plants. Differences among these isoforms in terms of their susceptibility to phosphinothricin (PPT), an analogue of glutamate and known inhibitor of GS, were found. The GS1 isoenzyme from MC was the most sensitive form, being inhibited by 50% at approximately 2.0 μM DL-PPT, whereas the GS2 from BSC presented the highest tolerance to the inhibitor (I₅₀=30 μM). The transferase-to-semibiosynthetic activity ratio for the MC isoforms, which was higher than the ratio for the BSC isoforms, and the differences shown by the isoforms in susceptibility to PPT predict important differences in the biochemical properties and regulation of GS isoenzymes. In this regard, the cytoplasmic isoenzymes, and especially the one in MC, due to its relatively high contribution to mesophyll cell GS activity, could play a vital role in nitrogen metabolism in maize. Received: 1 December 1999 / Revised: 7 February 2000 / Accepted: 23 February 2000     

Photodestruction of Chlorophyll inZea mays L. Leaves Under Different CO2 Concentration

Photodestruction of chlorophyll (Chl) inZea mays leaves, after their irradiation with high photon fluence rate (5000 μmol m⁻² s{−1}), was determined in fragments of whole leaves (WL) and also in fraction of mesophyll cells (MC) and bundle sheath cells (BSC) after their mechanical separation. The lag phase and the phase of photooxidation of Chl in MC chloroplasts were shorter than in BSC. Duration of both phases was reduced when the leaves were placed in 0 % CO₂ concentration in the atmosphere, while the increase of CO₂ concentration up to 0.3 % totally protected Chl against photodestruction in BSC within the 9 h experiment. During that period of time Chl was destructed by about 30 % in MC.     

The effect of cadmium on CO<Subscript>2</Subscript> exchange,variable fluorescence of chlorophyll,and the level of antioxidant enzymes in pea leaves

CO₂ exchange, variable chlorophyll fluorescence, the intensity of lipid peroxidation (POL), and the activity of antioxidant enzymes in leaves of two-week-old pea seedlings (Pisum sativum L.) exposed to 0.01, 0.1, and 1 mM aqueous solutions of Cd(NO₃)₂ for 2 h were studied. Incubation with Cd²⁺ ions resulted in a reduction of the maximum quantum efficiency of photosynthesis, CO₂ uptake rate, and photosystem II (PSII) activity, as assessed by F _v/F ₀ ratio. The intensity of POL in leaves of all treated seedlings was below or close to the control level. The activity of superoxide dismutase (SOD) and glutathione reductase (GR) increased in all treatments; the activity of ascorbate peroxidase (AP) exceeded the control level only in seedlings exposed to the high Cd²⁺ concentration (1 mM), and the activity of peroxidase increased at the low concentration (0.01 mM). We found that the reduction in the activity of the photosynthetic apparatus under conditions of 2-h-long Cd²⁺-induced stress was not related to an intensification of POL processes. It was concluded that stimulation of the activity of antioxidant enzymes—SOD, GR, AP, and peroxidase—is a pathway for pea plant adaptation to toxic effect of cadmium.Translated from Fiziologiya Rastenii, Vol. 52, No. 1, 2005, pp. 21–26.Original Russian Text Copyright © 2005 by Balakhnina, Kosobryukhov, Ivanov, Kreslavskii.     

Effect of temperature and photoperiod on efficiency of assimilated CO<Subscript>2</Subscript> conversion into the biomass of <Emphasis Type="Italic">Cucumis sativus</Emphasis>

The coefficient of effectiveness (K _e) of assimilated CO₂ conversion into dry matter of cucumber (Cucumis sativus L.) plants at the stage of four leaves as dependent on a photoperiod (8, 12, and 16 h) at an irradiance of 220 W/m² at the upper leaf level and the combinations of day and night temperatures: typical temperature of plant habitat (background temperature) of 25°C and heat- and cold-hardening temperatures (35 and 15°C, respectively) was determined in the multifactorial designed experiment. K _e reduced insignificantly at shortening of a photoperiod and greater at its lengthening. At background temperatures, K _e corresponded mainly to that of carbohydrate synthesis while the presence of cold-hardening temperatures in the thermoperiod increased K _e and heat-hardening temperature reduced it.Translated from Fiziologiya Rastenii, Vol. 52, No. 2, 2005, pp. 203–208.Original Russian Text Copyright © 2005 by Talanov, Popov, Kurets, Drozdov.This revised version was published online in April 2005 with a corrected cover date.     

Growth,photosynthesis, and metabolism of sugar beet at an early stage of exposure to elevated CO<Subscript>2</Subscript>

The effects of CO₂ concentration (C _a) on growth, photosynthesis, and the activity of enzymes associated with the translocation and assimilation of CO₂ were studied in sugar beet (Beta vulgaris L. subsp. saccharifera, cv. Ramonskaya) plants. The plants were grown in controlled-climate chamber to the stage of 3–4 leaves and then used in experiments. Experimental plants were exposed in boxes to doubled C _a (700 µl/l, 2C plants), whereas control plants were kept in a chamber with ambient atmosphere (350 µl/l, 1C plants). As compared with 1C plants, in 3 and 8 days, the leaf area of 2C plants increased by 14 and 26%, respectively. The rate of their photosynthesis (P _n) measured in 3, 6, and 8 days increased by 85, 47, and 52%, respectively, whereas in normal air, the values of P _n in 2C plants were by 12, 19, and 15% lower than in 1C plants. After 8-day growth, the content of soluble carbohydrates in the leaves of 2C plants attained 7.2%, being by 80% greater than in 1C plants; the content of starch did not exceed 3%. The total content of chlorophylls a and b in the leaves of 2C plants was by 14% greater than in 1C plants, but their ratio was essentially the same. The level of protein in 2C plants was by 13.4% lower than in 1C plants. The activity and content of Rubisco in 1C and 2C plants were similar. As compared with 1C plants, in 2C plants the activity of soluble carbonic anhydrase (sCA) was lower by 34% in 3 days and by 18% in 8 days; the activity of carbonic anhydrase of membrane preparations (mCA), was lower by 24 and 77%, respectively. Catalase activity in 2C plants became by 8% lower than in 1C plants only after 8 days. A reduction in the photosynthetic ability of 2C plants in ambient atmosphere, a decrease in activity of sCA and, especially, of mCA observed together with invariable activity and content of Rubisco in the leaf extracts are interpreted as early symptoms of acclimation of young plants of sugar beet to elevated CO₂.Translated from Fiziologiya Rastenii, Vol. 52, No. 2, 2005, pp. 184–190.Original Russian Text Copyright © 2005 by Ignatova, Novichkova, Mudrik, Lyubimov, Ivanov, Romanova.This revised version was published online in April 2005 with a corrected cover date.     

Identification and Characterization of Glycolate Oxidase and Related Enzymes from the Endocyanotic Alga Cyanophora paradoxa and from Pea Leaves

Glycolate oxidase (GO) has been identified in the endocyanom Cyanophora paradoxa which has peroxisome-like organelles and cyanelles instead of chloroplasts. The enzyme used or formed equimolar amounts of O₂ or H₂O₂ and glyoxylate, respectively. Aerobically, the enzyme did not reduce the artificial electron acceptor dichlorophenol indophenol. However, after an inhibitor of glycolate dehydrogenase, KCN (2 millimolar), was added to the assay medium, considerable aerobic glycolate:dichlorophenol indophenol reductase activity was detectable. The leaf GO inhibitor 2-hydroxybutynoate (30 micromolar), which binds irreversibly to the flavin moiety of the active site of leaf GO, inhibited Cyanophora GO and pea (Pisum sativum L.) GO to the same extent. This suggests that the active sites of both enzymes are similar. Cyanophora GO and pea GO cannot oxidize d-lactate. In contrast to GO from pea or other organisms, the affinity of Cyanophora GO for l-lactate is very low (K_m 25 millimolar). Another important difference is that Cyanophora GO produced sigmoidal kinetics with O₂ as varied substrate, whereas pea GO produced normal Michaelis-Menten kinetics. It is concluded that there is considerable inhomogeneity among the glycolate-oxidizing enzymes from Cyanophora, pea, and other organisms. The specific catalase activity in Cyanophora was only one-tenth of that in leaves. NADH-and NADPH-dependent hydroxypyruvate reductase (HPR) and glyoxylate reductase activities were detected in Cyanophora. NADH-HPR was markedly inhibited by hydroxypyruvate above 0.5 millimolar. Variable substrate inhibition was observed with glyoxylate in homogenates from different algal cultures. It is proposed that Cyanophora has multiple forms of HPR and glyoxylate reductase, but no enzyme clearly resembling leaf peroxisomal HPR was identified in these homogenates. Moreover, no serine:glyoxylate aminotransferase activity was detected. These results collectively indicate the possibility that the glycolate metabolism in Cyanophora deviates from that in leaves.     

Rates of glycolate synthesis and metabolism during photosynthesis of Euglena and microalgae grown on low CO2

The rate of glycolate excretion in Euglena gracilis Z and some microalgae grown at the atmospheric level of CO₂ was determined using amino-oxyacetate (AOA). The extracellular O₂ concentration was kept at 240 M by bubbling the incubation medium with air. Glycolate, the main excretion product, was excreted by Euglena at 6 mol·h^-1·(mg chlorophyll (Chl))^-1. Excretion depended on the presence of AOA, and was saturated at 1 mM AOA. A substituted oxime formed from glyoxylate and AOA was also excreted. Bicarbonate added at 0.1 mM did not prevent the excretion of glycolate. The excretion of glycolate increased with higher O₂ concentrations in the medium, and was competitively inhibited by much higher concentrations of bicarbonate. Aminooxyacetate also caused excretion of glycolate from the green algae, Chlorella pyrenoidosa, Scenedesmus obliquus and Chlamydomonas reinhardtii grown on air, at the rates of 2–7 mol·h^-1·(mg Chl)^-1 in the presence of 0.2–0.6 mM dissolved inorganic carbon, but the cyanobacterium, Anacystis nidulans, grown in the same way did not excrete glycolate. The efficiency of the CO₂-concentrating mechanism to suppress glycolate formation is discussed on the basis of the magnitude of glycolate formation in these low-CO₂-grown cells.Abbreviations AOA aminooxyacetate - Chl chlorophyll - DIC dissolved inorganic carbon - HPLC high-pressure liquid chromatography - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase This is the 16th paper in a series on the metabolism of glycolate in Euglena gracilis. The 15th paper is Yokota et al. (1985c)     

β-N-Oxalyl-L-α,β-Diaminopropionic Acid Protects the Activity of Glycolate Oxidase in Lathyrus sativus Seedlings under High Light

The content of -N-oxalyl-L-,-diaminopropionic acid (ODAP) and the activity of glycolate oxidase (GO) were positively correlated in the leaves of grass pea (Lathyrus sativus L.) seedlings. The activity of GO was kept at a steady level under the high light after treatment with ODAP. Although Na₂S can activate GO, it cannot maintain the activity of GO under the high light. The content of ODAP increased and the activity of GO decreased with increasing oxalate concentration used for seedling treatment. The GO activity was high enough to keep photosynthesis at a steady level under high light. These findings suggested that Lathyrus sativus, using oxalate as a precursor to produce ODAP, protected the GO activity at high irradiance by scavenging the hydroxyl radicals.     

Development of the Kranz structure during leaf growth in C4 Euphorbia maculate

The development of the Kranz structure was investigated in leaves of C₄ Euphorbia maculata using electron microscopy. Four leaf stages, i.e., primordial, immature, young, and mature, were examined, based on the photosynthetic tissue that surrounded the veins. The examination revealed how cells differentiated into distinct bundle sheath cells (BSCs) and mesophyll cells (MCs). Specialization of the BSCs was invariably associated with the development of the veins as well as the MCs. Precursors for BSC and MC were recognizable fairly early, at the immature stage, according to their position and differential enlargement Once these precursors were delimited from the procambial area, differentiation into each cell type occurred synchronously, in a coordinated manner. All cells enlarged as they were displaced from the Kranz precursor area, but the BSC precursors were initially larger and remained relatively larger than the other cell types throughout leaf development The developmental changes sharply distinguished BSCs from the adjacent MCs at the onset of Kranz formation and continued until maturity. Chloroplast enlargement also occurred during cell displacement, but the rate of enlargement was greater in BSCs, resulting in larger chloroplasts at later stages. However, no significant structural differences were detected among the chloroplasts of BSC and MC in the early stages. Most of the specialized features appeared at the young-leaf stage; structural dimorphism became prominent at the later stages. This enhanced development of the BSC chloroplasts was correlated with asymmetric distribution of cellular components. In addition, the BSC formed thin primary pit fields with numerous plasmodesmata. Peripheral reticulum was present, but generally was not conspicuous. We also discuss the characteristics of leaf anatomy and ultrastructure inE. maculata as they relate to the C₄ photosynthetic pathway.     

Isolation and Characterization of the ALP1 Protease from <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> and Its Protein Inhibitor from <Emphasis Type="Italic">Physarium polycephalum</Emphasis>

It is known that Aspergillus fumigatus secretes a serine protease ALP1 of the subtilisin family in the presence of extracellular protein substrates. We found conditions of A. fumigatus culturing that provide a high ALP1 activity inside cells without induction by extracellular proteins. The identity of the properties of the secreted and intracellular enzymes was shown. A thermostable protein inhibitor of the ALP1 protease was isolated from the plasmodium of myxomycete Physarum polycephalum. Its molecular mass is 32–33 kDa. It inhibits the ALP1 protease activity with IC₅₀ of 0.14 μM and was also shown to be a less efficient inhibitor of the activity of HIV-1 protease (IC₅₀ 2.5 μM).__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 3, 2005, pp. 259–268.Original Russian Text Copyright © 2005 by Davies, Kalinina, Samokhvalova, Malakhova, Scott, Venning, Volynskaya, Nesmeyanov.     

Subcellular Localization of Oxaloacetate Decarboxylase and Its Isolation from Maize Leaves

The activity of oxaloacetate decarboxylase was revealed in leaves of a C₄ plant, maize (Zea mays L.). This activity was unrelated to decarboxylase activities of other enzymes, e.g., NAD-malate dehydrogenase (EC 1.1.1.38) or NADP-malate dehydrogenase (EC 1.1.1.40), and was located in chloroplasts (83.1%). Using a four-step purification procedure, an electrophoretically pure enzyme preparation of oxaloacetate decarboxylase was obtained from maize leaves. The specific activity of the enzyme was 3.150 EU/mg protein, the factor of purification was 40.4, and the yield was 11.0%. The enzyme exhibited Michaelis–Menten kinetics with K _m for oxaloacetate 30 ± 5 M and pH optimum 7.1 ± 0.5. The metabolite-mediated regulation of oxaloacetate decarboxylase activity has been investigated. It is found that sodium chloride (1.0 mM) activates the enzyme, whereas ATP inhibits the enzyme activity.     

The Effects of Cold Acclimation of Winter Wheat Plants on Changes in CO<Subscript>2</Subscript> Exchange and Phenolic Compound Formation

We studied CO₂ exchange and phenolic compound production in various organs of unhardened and hardened winter wheat (Triticum aestivum L.) plants. The rates of CO₂ assimilation at saturating illumination (photosynthesis) and CO₂ evolution in darkness (respiration) declined substantially at the autumnal decrease of ambient temperature. However, because of a higher cold resistance of photosynthesis, the ratio of photosynthesis to respiration rates increased 1.5-fold. These gas exchange changes were accompanied by the accumulation of total soluble phenolics in leaves and a polymeric phenolic compound lignin in roots. We did not observe any changes in the production of either soluble or polymeric (lignin) phenolics in crowns.__________Translated from Fiziologiya Rastenii, Vol. 52, No. 3, 2005, pp. 366–371.Original Russian Text Copyright © 2005 by Zagoskina, Olenichenko, Klimov, Astakhova, Zhivukhina, Trunova.     

Sulphate uptake by leaf mesophyll and bundle sheath cells of maize plants

Uptake of³⁵S-sulphate by bundle sheath strands (BSC) from leaves of maize plants (Zea mays L. ev. Dekalb L 72 A) was higher than that by isolated mesophyll protoplasts (MC) of maize. Ion uptake followed the Michaelis-Menten kinetic satuiation curves. SO² ₄-uptake increased after addition of malate, NADPH, malate + NADP+ to BSC suspensions, but not to MC susp: nsions.     

The Effect of Various Alkaline Salts on the Glycolate Oxidase of Salicornia europaea and Pisum sativum in vitro

The response of glycolate oxidase from shoots of Salicornia europaea L. and from leaves of Pisum sativum L. to salt treatment during assay was studied by DCPIP reduction and O₂ uptake. In Pisum there was found up to five times more glycolate oxidase activity per gram fresh weight than in Salicornia. However, the calculation of the specific activity pointed out that this result was caused only by the high level of enzyme protein in Pisum, and that specific activity from both species was of equal size. By the DCPIP method it was shown that in test media containing up to 1.0 M NaCl or KCl glycolate oxidase of Salicornia was of equal size compared with the control (medium without additional salts). With 2.0 M NaCl or KCl the activity decreased by about 80 and 30% respectively. Glycolate oxidase of Pisum was somewhat more salt sensitive. 1.0 M NaCl or KCl reduced the activity by about 35%. In the presence of 2.0 M NaCl or KCl the enzyme activity from Pisum was inhibited to about 80 and 60% respectively. By substituting sulfates for chlorides the activity of glycolate oxidase from both Salicornia and Pisum was stimulated strongly. 1.5 M Na₂SO₄ and 0.5 M K₂SO₄ (both are saturated solutions) caused an increase of glycolate activity from Salicornia of about 225 and 185% respectively, and from Pisum of about 50 and 30% respectively. Studying the response of glycolate oxidase to salt treatment by O₂ uptake one must establish that with this method the degree of inhibition of enzyme activity at higher salt concentrations was always more severe than with dye reduction. Addition of 1.0 M NaCl or KCl to the assay medium caused an inhibition of glycolate oxidase activity from Salicornia of about 50% and from Pisum of about 60%. 2.0 M NaCl or KCl reduced the enzyme activity of both Salicornia and Pisum to nearly 10% of control activity. Furthermore, in contrast to DCPIP reduction no stimulating effect of sulfates on glycolate oxidase activity was detectable. Indeed, the inhibitory effect of sulfates was very slight. 1.0 M Na₂SO₄ caused a mean inhibition of glycolate oxidase activity of only 15% with both species, and in the presence of 1.5 M Na₂SO₄ 50% of control activity was measured. At maximal K₂SO₄ concentrations (0.5 M) glycolate oxidase from both Salicornia and Pisum was also unaffected. It is supposed that the described salt tolerance of glycolate oxidase in vitro, possibly is due to an adaptation of the enzyme to high salt levels within peroxisomes in vivo.     

C4 syndrome of the species in theDichotomiflora group of the genusPanicum (Gramineae)

Leaf anatomy, pattern of post-illumination CO₂ burst (PIB) and activity of three C₄-acid decarboxylating enzymes in C₄ photosynthesis were investigated with the leaves of five species in theDichotomiflora group of the genusPanicum. All species had mestome sheaths, exhibited the sharp pattern of PIB in less than 30 sec of darkness and were classified as NAD-malie enzyme species biochemically. However, they clearly fell into two groups according to the difference in chloroplast location in bundle sheath cells (BSC).P. coloratum var.makarikariense, P. lanipes andP. stapfianum had centripetal chloroplasts, whereasP. laevifolium andP. longijubatum had centrifugal chloroplasts, whereas cv. Kabulabula and cv. Solai had centrifugal chlorplasts. The results indicate that theDichotomiflora group had the two leaf anatomical variations of NAD-malic enzyme species. In addition, the results onP. coloratum suggest that this species may be divided into two separate species by chloroplast location in BSC. The ultrastructural features of leaves ofP. dichtomiflorum, NAD-malic enzyme species with centrifugal chloroplasts, were also investigated. Chloroplasts in BSC had well-developed grana, and numerous large mitochondria with extensively developed internal membrane structure were restricted to the area between the chloroplsts and the vacuole in BSC.     

Catalase activity and rhythmic patterns in mouse brain, kidney and liver

To protect tissues from damaging effects of reactive oxygen species (ROS), organisms possess enzymatic and non-enzymatic antioxidant systems. Cytosolic-enzyme catalase (CAT) is a component of the antioxidant defence system that reduces hydrogen peroxide (H₂O₂) to water (H₂O). The aim of this study was to assess the variation of antioxidant enzyme CAT activity in brain, kidney and liver of adult male mice according to tissue-specific and temporal patterns within a 24-h period (12:12 L/D). The CAT activity was assayed at 4-h intervals. The Cosinor test programme was used to detect and confirm the best corresponding rhythm. In liver, the circadian rhythm of CAT was associated with ultradian components. The prominent circadian rhythm (with a period τ = 24 h) showed a peak located at the middle of the dark phase, more precisely  17 HALO (Hours After Light Onset). In kidney, only a circadian rhythm of CAT was validated with a peak time located at  17 HALO. However, in brain, the time pattern of CAT activity showed two peak times at  1 and  17 HALO, illustrating the existence of an ultradian rhythm (with a period τ = 12 h). The results showed significant organ differences with the highest activity in liver, compared with kidney (− 89%) and brain (− 98%). This might be related to several factors such as their respective physiological function, the risk of exposure to oxidative damage and the balance between synthesis and degradation of proteins during “normal metabolism”. Moreover, CAT activity revealed differences in time-related changes across a 24-h period that were more obvious in peak levels between the three tissues.