Effects of dark preincubation and chloramphenicol on blue light-induced CO2 incorporation in Chlorella cells

Chlorella cells incubated in the dark longer than 12 hr showedpronounced blue light-induced ¹⁴CO₂ fixation into aspartate,glutamate, malate and fumarate (blue light effect), whereasthose kept under continuous light showed only a slight bluelight effect, if any. 2) During dark incubation of Chlorellacells, phosphoenolpyruvate carboxylase activity and the capacityfor dark ¹⁴CO₂ fixation decreased significantly, whereas ribulose-1,5-diphosphatecarboxylase activity and the capacity for photosynthetic ¹⁴CO₂fixation (measured under illumination of white light at a highlight intensity) did not decrease. 3) In cells preincubatedin the dark, intracellular levels of phosphoenolpyruvate and3-phosphoglycerate determined during illumination with bluelight were practically equal to levels determined during illuminationwith red light. 4) The blue light effect was not observed incells incubated widi chloramphenicol, indicating that blue light-inducedprotein synthesis is involved in the mechanism of the effect. (Received April 9, 1971; )     

Phosphoenolpyruvate Carboxylase from Heterotrophically Cultured Catharanthus roseus Cells

Maximum activity of phosphoenolpyruvate carboxylase (PEPC, EC4.1.1.31) was detected at the stationary phase of growth ofCatharanthus roseus cells in a heterotrophic culture. The activityof PEPC, after partial purification by fractionation with ammoniumsulphate and chromatography on Q-Sepharose, was greatly influencedby pH. The K_m of phosphoenolpyruvate (PEP) was 23 µM atpH 8·0 and 45 µM at pH 7·4. Malate, aspartate,citrate, ATP, pyrophosphate and Pi acted as inhibitors of PEPC,but the extent of inhibition varied in each case with the pHof the reaction mixture. By contrast, glucose-6-phosphate, fructose-1,6-bisphosphateand acetyl-CoA, known as stimulators of the activity of PEPCfrom other sources, had little or no effect on the activityof the partially purified PEPC. The possible role and mechanismof regulation of PEPC in C. roseus cells are discussed.Copyright1994, 1999 Academic Press Catharanthus roseus, Apocynaceae, Madagascar periwinkle, suspension culture, phosphoenolpyruvate carboxylase, enzyme kinetics, glycolysis     

Activities of enzymes concerned with pyruvate and oxaloacetate metabolism in the heart and liver of developing sheep.

1. In order to assess whether the potential ability of heart ventricular muscle and liver to metabolise substrates such as alanine, aspartate and lactate varies as the sheep matures and its nutrition changes, the activities of the following enzymes were determined in tissues of lambs obtained at varying intervals between 50 days after conception to 16 weeks after birth and in livers from adult pregnant ewes: lactate dehydrogenase (EC 1.1.1.27), alanine aminotransferase (EC 2.6.1.2), pyruvate kinase (EC 2.7.1.40), pyruvate carboxylase (EC 6.4.1.1), phosphoenolpyruvate carboxykinase (GTP)(EC 4.1.1.32), malate dehydrogenase (EC 1.1.1.37), aspartate aminotransferase (EC 2.6.1.1) and citrate (si)-synthase (EC 4.1.3.7). 2. In the heart a most marked increase in alanine aminotransferase activity was found throughout development. During this period the activities of citrate (si)-synthase, lactate dehydrogenase and pyruvate carboxylase also increased. There were no substantial changes in the activities of aspartate aminotransferase, malate dehydrogenase or pyruvate kinase. Pyruvate kinase activities were five times greater in the heart compared with those found in the liver. No significant activity of phosphoenolpyruvate carboxykinase (GTP) was detected in heart muscle. 3. In the liver the activities of both alanine aminotransferase and aspartate aminotransferase increased immediately following birth although the activity of alanine aminotransferase was lower in livers of pregnant ewes than in any of the lambs. As with alanine aminotransferase the highest activities of lactate dehydrogenase were found during the period of postnatal growth. No marked changes were observed in malate dehydrogenase or citrate (si)-synthase activities during development. A small decline in pyruvate kinase activity occurred whilst the activities of pyruvate carboxylase and phosphoenolpyruvate carboxykinase (GTP) tended to rise during development.     

Photosynthetic metabolism in the cyanophytaOscillatoria rubescens D.C.

Short term¹⁴C labelling experiments and enzymatic activities related to primary pathways of photosynthesis have been studied in the cyanophytaOscillatoria rubescens D.C. from axenic cyclostat cultures. Responses of samples from cultures with different amounts of nitrogen are presented and compared. Variations in photosynthetic pigments are used to quantify the degree of nitrogen starvation at different levels.PEPcarboxylase activity remains low and is not affected by nitrogen starvation. RuBPcarboxylase activity is lowered to nearly two thirds of its normal metabolic rate by starvation but PEPcarboxykinase and aspartate aminotransferase activities are significantly higher in this case. Malate dehydrogenase is slightly altered and malic enzyme is never active. Starved algae replaced in fresh complete media fix rapidly¹⁴C in nitrogen compounds such as amino acids. Results are discussed in regard to both physiological and ecological characteristics ofO. rubescens. PEPcarboxykinase can play a role in making efficient use of HCO ₃ ^- .Abbreviations AAT aspartate aminotransferase - APC allophycocyanin - ASM 1 algal synthetic medium, 1st modification - DW dry weight - GAPDH glyceraldehyde 3-phosphate dehydrogenase - MDH malate dehydrogenase - PC phycocyanin - PE phycoerythrin - PEP phophoenolpyruvate - PEPC phosphoenolpyruvate carboxylase - PEPCK phophoenolpyruvate carboxykinase - PGA 3-phosphoglyceric acid - RuBPC ribulose bisphosphate carboxylase     

The relationship between mitochondrial heterogeneity and gluconeogenesis in liver mitochondria of the rat, pigeon and guinea pig.

Isolated mitochondria of pigeon and guinea pig liver were subjected to zonal centrifugation. With pigeon liver mitochondria there was uniform distribution of pyruvate carboxylase, phosphoenolpyruvate carboxykinase, malate dehydrogenase, aspartate aminotransferase and glutamate dehydrogenase activities. Guinea pig liver mitochondria demonstrated two pyruvate carboxylase and phosphoenolpyruvate carboxykinase maxima but only one maximum with aspartate aminotransferase, malate dehydrogenase and glutamate dehydrogenase. Mitochondrial enzyme levels in rat, pigeon and guinea pig indicate different roles of certain gluconeogenic enzymes in the transport of carbon and hydrogen in and out of mitochondria.     

Effect of LED light spectra on starvation-induced oxidative stress in the cinnamon clownfish Amphiprion melanopus

The present study aimed to test starvation-induced oxidative stress in the cinnamon clownfish Amphiprion melanopus illuminated by light-emitting diodes (LEDs): red (peak at 630nm), green (peak at 530nm), and blue (peak at 450nm) within a visible light. We investigated the oxidative stress induced by starvation for 12days during illumination with 3 LED light spectra through measuring antioxidant enzyme (superoxide dismutase [SOD] and catalase [CAT]) mRNA expression and activity; CAT western blotting; and measuring lipid peroxidation [LPO]), plasma H(2)O(2), lysozyme, glucose, alanine aminotransferase (AlaAT), aspartate aminotransferase (AspAT), and melatonin levels. In green and blue lights, expression and activity of antioxidant enzyme mRNA were significantly lower than those of other light spectra, results that are in agreement with CAT protein expression level by western blot analysis. Also, in green and blue lights, plasma H(2)O(2), lysozyme, glucose, AlaAT, AspAT, and melatonin levels were significantly lower than those in other light spectra. These results indicate that green and blue LEDs inhibit oxidative stress and enhance immune function in starved cinnamon clownfish.     

Interactions of Phosphoenolpyruvate Carboxylase and Pyruvic Kinase in Developing Soybean Seeds

Developing soybean seeds contain phosphoenolpyruvate (PEP) carboxylase,pyruvic kinase, malate dehydrogenase, aspartate aminotransferase,alanine aminotransferase and malic enzyme activities. PEP carboxylasemay be important in competing with pyruvic kinase and directinga portion of glycolytic carbon towards oxaloacetate synthesis.The oxaloacetate can then be converted to aspartate and malate.Malic enzyme produces pyruvate and NADPH from malate, and thismay be an important additional source of reducing power forlipid biosynthesis. In the presence of high levels of PEP carboxylaseit is possible to demonstrate PEP formation by pyruvic kinase.PEP carboxylase and pyruvic kinase independently compete forPEP in a mixed system. Soybean seed extracts readily convertedradioactive PEP into alanine and aspartate when supplementedwith ADP, Mg²⁺, K+, HCO₃– and glutamate. Under varyingconditions of pH, metal ions, PEP, enzyme concentration andtime both alanine and aspartate were always produced. Possiblythe final products of glycolysis should be considered as pyruvateand oxaloacetate in plants. (Received April 22, 1981; Accepted June 26, 1981)     

The Regulation of the Starch-Malate Balances during Volume Changes of Guard Cell Protoplasts

The enzymatic activities of phosphoenolpyruvate carboxylase(EC 4.1.1.31 [EC] ), ‘malic enzyme’ (EC 1.1.1.40 [EC] ), phosphofmctokinase(EC 2.7.1.11 [EC] ) and fructosebisphosphatase (EC 3.1.3.11 [EC] ) weremeasured during the swelling and shrinking of isolated and purifiedguard cell protoplasts (Vicia faba) in darkness. The volumeincrease was accompanied by the activation of phosphofructokinaseand a short stimulation of phosphoenolpyruvate carboxylase,at the same time the ‘malic enzyme’ and fructosebisphosphatasewere inhibited. However, during the shrinkage of guard cellprotoplasts these two enzymes were activated in contrast tophosphoenolpyruvate carboxylase and phospho-fructokinase. Becauseof the dramatic increase of phosphoenolpyruvate carboxylaseactivity during the swelling, this enzyme was assumed to actas a trigger for the swelling phase.     

Photochemical Apparatus Organization in the Diatom Cylindrotheca fusiformis: Photosystem Stoichiometry and Excitation Distribution in Cells Grown under High and Low Irradiance

The photochemical apparatus organization in the thylakoid membraneof the diatom Cylindrotheca fusiformis was investigated in cellsgrown under high and low irradiance. High light (HL, 200µE.m^–2.s^–1)grown cells displayed a relatively low fucoxanthin to chlorophyll(Chl) ratio, a low photosystem (PS) stoichiometry (PSII/PS I=1.3/1.0)and a smaller photosynthetic unit size in both PS I and PS II.Low light (LL, 30µE.m^–2.s^–1) grown cells displayeda 30% elevated fucoxanthin content, elevated PS II/PS I=3.9/1.0and larger photosynthetic unit size for PS II (a change of about100%) and for PS I (by about 30%). In agreement, SDS polyacrylamidegel electrophoresis of thylakoid membrane polypeptides showedgreater abundance of PS I, RuBP carboxylase and ATP synthasepolypeptides in HL cells. In contrast, LL grown cells exhibitedgreater abundance of light-harvesting complex polypeptides.Assuming an efficiency of red (670 nm) light utilization of1.0, the measured efficiency of blue (481 nm) light utilizationwas 0.64 (HL cells) and 0.72 (LL cells). The lower efficiencyof blue versus red light utilization is attributed to the quenchingof absorbed energy by non-fucoxanthin carotenoids. Differencesin the efficiency of blue light utilization between HL and LLgrown cells are attributed to the variable content of fucoxanthin.The results support the hypothesis of a variable Chl a-Chl c-fucoxanthinlight-harvesting antenna associated with PS II and PS I in Cylindrotheca. (Received February 10, 1988; Accepted April 6, 1988)     

No Light Activation and High Malate Sensitivity of Phosphoenolpyruvate Carboxylase in Guard Cell Protoplasts of Commelina communis L.

Guard cell protoplasts (GCP) were prepared from leaves of Commelinacommunis L. and phosphoenolpyruvate carboxylase (PEPc) activityrecorded after injection of the protoplasts directly into theassay medium. The GCP were lysed immediately by the presenceof Triton X-100 and a lowered osmotic concentration in the assaycuvette enabling PEPc activity to be measured with ‘nascent’enzyme. There was no light activation of the enzyme with K_mPEP (about 3.7 mol m^–3) and Vmax being similar in light-ordark-treated protoplasts. Illumination of the GCP in the presenceof CO₂-free air and KCI, a treatment which is known to swellGCP, did not change the kinetics. PEPc activity at saturating PEP was very sensitive to malateinhibition, 20 mmol m^–3 (the I₅₀ value) inhibiting activityby about 50%. Inhibition was similar in light- or dark-treatedprotoplasts. Malate inhibition was, however, much less (I₅₀= 500 mmol m^–3) if the enzyme source was a protoplastextract kept in the absence of glycerol. Inclusion of 20% glycerolin the extraction medium maintained the enzyme in the malate-sensitiveform as occurred in the in vivo assays. The high apparent K_mPEP and the high sensitivity to malate inhibition of GCP PEPcare features unlike those observed with PEPc from leaf tissuesof C₄ and CAM plants and from GCP extracts. PEPc activity increased slightly in the presence of KCI in theassay medium up to about 10 mol m^–3 and thereafter activityslowly declined as KCI concentrations increased further. Key words: Guard cell protoplasts, phosphoenolpyruvate carboxylase     

Purification of NADP-Malic Enzyme and Phosphoenolpyruvate Carboxylase from Sugar Cane Leaves

A procedure is described for the purification of phosphoenolpyruvatecarboxylase (EC 4.1.1.31 [EC] ) and NADP-dependent malic enzyme (EC1.1.1.40 [EC] ) from sugar cane leaves. Each enzyme was purified tohomogeneity as judged by sodium dodecyl sulfate-polyacrylamidegel electro-phoresis, with about 30% yield. Phosphoenolpyruvatecarboxylase was purified 54-fold. A molecular weight of 400,000and a homotetrameric structure were determined for the nativeenzyme. The purified carboxylase had a specific activity of20.0 {diaeresis}mol (mg protein)_–1 min_–1, and wasactivated by glucose-6-phosphate and inhibited by L-malate.K_m values at pH 8.0 for phosphoenolpyruvate and bicarbonatewere 0.25 and O.l0 mM, respectively. NADP-malic enzyme, 356-foldpurified, exhibited a specific activity of 71.2 {diaeresis}mol(mg protein)_–1 min_–1 and was characterized as ahomotetramer with native molecular weight of 250,000. Purifiedmalic enzyme showed an absolute specificity for NADP⁺ and requireda divalent metal ion for activity. K_m values of 0.33 and 0.008mM for L-malate and NADP⁺, respectively, were determined. Thisenzyme was inhibited by several organic acids, including ketoand amino acids; while succinate and citrate increased the enzymeactivity when assayed with 10{diaeresis}M L-malate. The effectsshown by amino acids and by citrate were dependent on pH, beinghigher at pH 8.0 than at pH 7.0. (Received October 26, 1988; Accepted February 3, 1989)     

Change in Properties of Phosphoenolpyruvate Carboxylase in Kalanchoe daigremontiana with Leaf Age

The activity of phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31 [EC] )increased with leaf age in Kalanchoe daigremontiana. Polyacrylamidegel electrophoresis showed 3 bands of PEP carboxylase activity,one predominant in young leaves, another predominant in olderleaves. PEP carboxylase activities in desalted extract differedaccording to pH, at acidic pH the enzyme activity of young leavesdecreased drastically, whereas that of older leaves remainednearly constant. (Received August 2, 1982; Accepted September 27, 1982)     

Synthesis and degradation of phosphoenolpyruvate carboxylase in rat liver and adipose tissue. Changes during a starvation-re-feeding cycle

A specific antibody against liver cytosol phosphoenolpyruvate carboxylase (EC 4.1.1.32) was used to isolate the enzyme from liver and adipose tissue. With this technique we have shown that phosphoenolpyruvate carboxylase synthesis in starved rats accounts for 3% of the total synthesis of cytosol protein in each tissue. Re-feeding starved animals decreases this relative rate of phosphoenolpyruvate carboxylase synthesis to 0.2% and 1% respectively in liver and adipose tissue, and the activity of the enzyme in each tissue is decreased to 25% of the starvation value. An additional starvation period is accompanied by an increased rate of enzyme synthesis, but the response to starvation is considerably slower than that caused by re-feeding. The degradation rate of phosphoenolpyruvate carboxylase is also subject to regulation. Thus re-feeding starved animals decreases the half-life of the enzyme in liver from 13h to 5.2h, but the rapid rate of degradation is maintained at least during the first 20h of subsequent starvation. Only slight changes in the degradation rate of phosphoenolpyruvate carboxylase are found in adipose tissue. We conclude that the large alterations in the rate of enzyme synthesis during a starvation–re-feeding cycle are the major cause of fluctuations in activity.     

Some Observations on a Circadian Rhythm in Carbon Dioxide Compensation in Bryophyllum fedtschenkoi

Detached shoots of Bryophyllum fedtschenkoi maintained in continuouslight and at a constant temperature exhibit a circadian rhythmin CO₂ compensation. The rhythm has a period of 21.6±0.1h at 23 °C and its phase can be set and shifted by suitabletreatments. These observations suggest that the ryhthm is trulyendogenous in nature. The phase is set by a light-on stimulus, or a drop in lightintensity. Phase shift can be induced by short periods (3 h)of reduced light intensity as well as similar periods of darknessgiven during the day phase of the rhythm. A change from whitelight to the same number of incident quanta of monochromaticlight of 450 nm (blue), 550 nm (green), or 652 nm (red) alwaysshifted the phase, but the degree of shift was less in red lightthan in blue or green. The plant reacted to achange to blueor green light as though it had been placed in the dark. Althbughred light was apparently ‘recognized’, the changefrom white to red was still sufficient to alter the phase ofthe rhythm. The possible role of phosphoenolpyruvate carboxylase activityin controlling the rhythm is discussed.     

Blue Light-Induced In Vivo Absorbance Changes and In Vitro Activation of Nitrate Reductase in a Nitrate-Starved Chlorella Mutant

Illuminating a colorless mutant of Chlorella vulgaris 11h (M125)with blue light caused a reversible photoreduction of b-typecytochrome, i.e., absorbance increases at 423, 525 and 557 nm.This light-induced reduction of cytochrome b was most pronouncedin nitrate-starved cells, which showed some blue light responsesin carbon metabolism, including enhancement of respiration byblue light as reported previously. Prolonged illumination withblue light caused a decrease in the rate of the reduction. The photoactivation of nitrate reductase in the mutant cellswas studied in both cell-free crude extract and purified enzyme.The absorption spectrum of purified enzyme showed three peaksat 423, 525 and 557 nm after the addition of a reductant, indicatingthat the spectrum is that of cytochrome b associated with nitratereductase. Nitrate reductase activity was easily enhanced byblue light illumination after 1 min; red light had no effecton it. The blue light activation of nitrate reductase was notsignificant in growing cells, which showed its high activity. The relationship between the blue light-induced reduction ofcytochrome b and carbon metabolism is discussed. (Received September 30, 1987; Accepted February 9, 1988)     

Illumination increases the phosphorylation state of maize leaf phosphoenolpyruvate carboxylase by causing an increase in the activity of a protein kinase

Illumination of maize leaves increases the phosphorylation state of phosphoenolpyruvate carboxylase and reduces the sensitivity of the enzyme to feedback inhibition by malate. Red, white and blue light were each found to be equally potent, and the effect of light was blocked by 3(3,4-dichlorophenyl)-1,1-dimethylurea. A phosphoenolpyruvate carboxylase kinase was partially purified from illuminated maize leaves by a three-step procedure. Phosphorylation of phosphoenolpyruvate carboxylase by this protein kinase reached 0.7-0.8 molecules/subunit and correlated with a 3- to 4-fold increase in Ki for malate. The protein kinase was inhibited by L-malate, but was insensitive to a number of other potential regulators. Freshly prepared and desalted extracts of darkened maize leaves contained very little kinase activity, but the activity appeared when leaves were illuminated for 30-60 min before extraction. The catalytic subunit of protein phosphatase 2A from rabbit skeletal muscle, but not that of protein phosphatase 1, could dephosphorylate phosphoenolpyruvate carboxylase. The protein phosphatases 1 and 2A activities of maize leaves were not affected by illumination. It is suggested that the major means by which light stimulates the phosphorylation of phosphoenolpyruvate carboxylase is by an increase in the activity of the protein kinase.     

DEVELOPMENT OF MITOCHONDRIAL PYRUVATE METABOLISM IN RAT BRAIN

The activities of a number of mitochondrial enzymes involved in the metabolism of pyruvate during development of the rat brain were investigated. The rates of decarboxylation of [1-¹⁴C]pyruvate to ¹⁴CO₂ via pyruvate dehydrogenase and the fixation of H¹⁴CO₃^? in the presence of pyruvate via pyruvate carboxylase by brain homogenates were very low in newborn rats. These rates increased markedly by about four-fold and 15-fold respectively during 10–35 postnatal days. The rates of the fixation of H¹⁴CO₃^? by cerebral homogenates were supported by the development of the activity of pyruvate carboxylase in rat brain. The activities of citrate synthase, aconitase, NAD-malate dehydrogenase, aspartate aminotransferase, alanine aminotransferase and phosphoenol-pyruvate carboxykinase were very low in the particulate fraction of the newborn rat brain. The activities of all these enzymes increased makedly by about three- to 10-fold during 10–35 days after birth. The activity of mitochondrial phosphoenolpyruvate carboxykinase from rat brain was not precipitated by an antibody prepared against rat liver cytosolic phosphoenolpyruvate carboxykinase suggesting that cerebral mitochondrial enzyme is immunologically different from that of the cytosolic form in hepatocytes. The significance of the development of the cerebral mitochondrial metabolism is discussed in relation to biochemical maturation of the brain.     

Phosphate Deficiency in Maize. II. Enzyme Activities

Low-phosphate (P) treatment decreased photosynthetic rates ofmaize plants (Zea mays L.) by about 50% 18 to 19 days afterplanting [Usuda and Shimogawara (1991) Plant Cell Physiol. 32:497]. Low-P treatment decreased the enzyme activities differentially(by 0-49%). The significance of the decreased activities ofpyruvate.Pj dikinase (by 29%), phosphoenolpyruvate carboxylase(by 49%), and ribulose 1,5-bisphosphate carboxylase (by 42%)in the detrimental effects of low-P treatments on the ratesof photosynthesis is discussed. (Received August 9, 1991; Accepted October 1, 1991)     

Some Biochemical Characteristics of Guard Cell and Mesophyll Cell Protoplasts from Commelina communis L.

Guard cell and mesophyll cell protoplasts of Commelina communisL., were isolated and used to investigate their various biochemicalcharacteristics. Contamination of the samples by other celltypes was very low and viability of the protoplasts, assessedby the use of neutral red, Evans blue and fluorescein diacetate,was high (89–98%). Mesophyll cell protoplasts containedmore chlorophyll (x 47), more soluble protein (x 10), more totalN (x 36) and more DNA (x 9) than guard cell protoplasts. Theabsorption spectra of protoplast extracts were similar for bothcell types except that below 400 nm there was a large increasein absorption by the guard cell protoplast extract. In guardcell protoplast extracts, high levels of activity of phosphoenolpyruvatecarboxylase (E.C. 4.1.1.31 [EC] ), NAD malate dehydrogenase (E.C.1.1,1.37), NADP malic enzyme (E.C. 1.1.1.40 [EC] ) and carbonic anhydrase(E.C. 4.2.1.1 [EC] ) were detected while only low levels of pyruvate-orthophosphatedikinase (E.C. 2.7.9.1 [EC] ) activity were detected. Glycollate oxidase(E.C. 1.1.3.1 [EC] ), ribulose-l,5-bisphosphate carboxylase (E.C 4.1.1.39 [EC] ),NADP malate dehydrogenase (E.C. 1.1.1.82 [EC] ) and NAD malic enzyme(E.C. 1.1.1.39 [EC] ) were not detected in guard cell protoplast extracts.High levels of ribulose-1, 5-bisphosphate carboxylase, glycollateoxidase, NAD malate dehydrogenase and carbonic anhydrase weredetected in mesophyll cell protoplast extracts which is typicalof C₃ plants. A pathway of carbon flow during stomatal openingand closing is proposed. Key words: Carbon metabolism, Commelina communis, guard cell protoplasts, mesophyll cell protoplasts, stomata