Comparative Studies of NAD-Malic Enzyme from Leaves of Various C4 Plants

Using butyl-TSK-gel chromatography, we purified NAD-malic enzyme(ME) (EC 1.1.1.39 [EC] ), which is involved in C₄ photosynthesis,to electrophoretic homogeneity, from leaves of Amaran-thus tricolor.Molecular weights of the native and SDS-denatured enzyme fromA. tricolor were 490 kDa and 61 kDa, respectively. During assayof the enzyme there was a slow reaction transient in the formof a lag before a steady-state rate was reached. The durationof this lag was inversely proportional to the concentrationof each substrate and the activator, fructose- 1,6-bis-phosphate(FBP). The optimal pH of the reaction fell with decreasing concentrationsof either malate or FBP. High pH prolonged the lag in reaction. Double reciprocal plots of the enzymatic activity as a functionof the concentration of malate yielded straight lines and didnot show any cooperativity for binding of malate. The enzymefrom A. tricolor was not inhibited by either HCO^–₃ orCO₂. At different concentrations of malate, the nature of theactivating effect of FBP was compared among the purified enzymesfrom A. tricolor and the C₄ monocots Eleusine coracana and Panicumdichotomiflorum. At low levels of malate, FBP markedly stimulatedthe enzyme from each species. In contrast, at saturating levelsof malate, the response of enzymes to increasing concentrationsof FBP was different and depended on the source of enzyme. The immunochemical properties of the enzymes from the threespecies were compared using an enzyme-linked immunoadsorbentassay with antisera raised against the purified enzymes fromthe three species. Different cross-reactivities were observedamong the enzymes from different sources. The N-terminal aminoacid sequences of NAD-MEs from the three species were determinedand some differences were found among the three enzymes. ²Permanent address; Tohoku National Agricultural ExperimentStation, Morioka, 020-01 Japan. ³Permanent address; National Grassland Research Institute, Nishinasuno,Tochigi, 329-27 Japan. (Received December 12, 1988; Accepted February 17, 1989)     

Sodium Requirement of Monocotyledonous C4 Plants for Growth and Nitrate Reductase Activity

The effects of Na application on growth and nitrate reductaseactivity of seven C₄ plant species, Zea mays, Echinochloa crus-galli,Panicum miliaceum, Panicum coloratum, Panicum dichotomiflorum,Panicum maximum and Chloris gayana were studied. Except forZ. mays and P. miliaceum, Na application enhanced growth significantly,and concurrent increases in nitrate reductase activities weredetected in Panicum coloratum, Panicum dichotomiflorum, Panicummaximum and Chloris gayana. ¹Present address: International Research Institute, Ciba GeigyJapan Ltd., Takarazuka, Hyogo 665, Japan. ²Present address: Photobiology Lab., Research Institute forFood Science, Kyoto Univ., Uji, Kyoto 611, Japan. (Received May 2, 1988; Accepted August 22, 1988)     

Characterization of a Maize Ca2+-Dependent Protein Kinase Phosphorylating Phosphoenolpyruvate Carboxylase

Phosphoenolpyruvate carboxylase (PEPC) [EC 4.1.1.31 [EC] ] of plantsundergoes regulatory phosphorylation in response to light ornutritional conditions. However, the nature of protein kinase(s)for this phosphorylation has not yet been fully elucidated.We separated a Ca²⁺-requiring protein kinase from Ca²⁺-independentone, both of which can phosphorylate maize leaf PEPC and characterizedthe former kinase after partial purification. Several linesof evidence indicated that the kinase is one of the characteristicCa²⁺-dependent but calmodulin-independent protein kinase (CDPK).Although the M_r, of native CDPK was estimated to be about 100kDa by gel permeation chromatography, in situ phosphorylationassay of CDPK in a SDS-polyacrylamide gel revealed that thesubunit has an M_r of about 50 kDa suggesting dimer formationor association with other protein(s). Several kinetic parameterswere also obtained using PEPC as a substrate. Although the CDPKshowed an ability of regulatory phosphorylation (Ser-15 in maizePEPC), no significant desensitization to feedback inhibitor,malate, could be observed presumably due to low extent of phosphorylation.The kinase was not specific to PEPC but phosphorylated a varietyof synthetic peptides. The possible physiological role of thiskinase was discussed. ¹Present address: NEOS Central Research Laboratory, 1-1 Ohike-machi,Kosei-cho, Shiga, 520-3213 Japan. ²Present address: Chugai Pharmaceutical Co., Ltd., 1-135 Komakado,Gotemba, 412-0038 Japan. ⁴N.O. and N.Y. contributed equally to this work.     

Cloning, Expression, and Characterization of a Root-Form Phosphoenolpyruvate Carboxylase from Zea mays: Comparison with the C4-Form Enzyme

A full-length cDNA for maize root-form phosphoenolpyruvate carboxylase(PEPC) was isolated. In the coding region, the root-form PEPCshowed 76 and 77% identity with the C₄- and C₃-form PEPCs ofmaize, respectively, at the nucleotide level. At the amino acidlevel, the root-form was 81 and 85% identical to the C₄- andC₃-form PEPCs, respectively. The entire coding region was insertedinto a pET32a expression vector so that it was expressed underthe control of T7 promoter. The purified recombinant root-formPEPC had a V_max value of about 28 mol min^–1(mg protein)¹at pH 8.0. The K_m values of root-form PEPC for PEP and Mg²⁺were one-tenth or less of those of C₄-form PEPC when assayedat either pH 7.3 or 8.0, while the value for HCO^–₃ wasabout one-half of that of C₄-form PEPC at pH 8.0. Glucose 6-phosphateand glycine had little effect on the root-form PEPC at pH 7.3;they caused two-fold activation of the C₄-form PEPC. The K_i(L-malate) values at pH 7.3 were 0.12 and 0.43 raM for the root-and C₄-form PEPCs, respectively. Comparison of hydropathy profilesamong the maize PEPC isoforms suggested that several stretchesof amino acid sequences may contribute in some way to theircharacteristic kinetic properties. The root-form PEPC was phosphorylatedby both mammalian cAMP-dependent protein kinase and maize leafprotein kinase, and the phosphorylated enzyme was less sensitiveto L-malate. ¹These authors contributed equally to this work. ²Present address: Otsuka Chemical Co. Ltd., 463 Kagasuno, Kawauchi-cho,Tokushima, 771-0130 Japan. ³Present address: Sumitomo Pharmaceuticals Research Center,1-98, Kasugade, Naka 3-cho-me, Konohana-ku, Osaka, 554-0022Japan.     

Some properties of shikimate: NADP oxidoreductase produced in sweet potato root tissue after slicing

The activity of shikimate: NADP oxidoreductase [EC 1. 1. 1.25] in sweet potato root tissue increased soon after slicing.Enzyme preparations obtained from both sliced tissue and fromfresh tissue probably contained a single enzyme component, andthey showed identical chromatographical behaviour. K_m values of the enzyme for NADP and shikimate were 1.0x10^–4Mand 1.3 x 10^–3M, respectively. Enzyme activity was potentlyinhibited by SH-inhibitors such as p-chloromercuribenzoate andoxidized glutathione. Enzyme activity was affected neither by mononucleotides suchas ATP, ADP and AMP, divalent cations, Mg⁺⁺, Ca⁺⁺ and Mn⁺⁺,nor by metabolites such as tryptophan, phenylalanine, tyrosineand t-cinnamic acid which are involved in aromatic compoundsyntheses. The enzyme rapidly lost its activity. This inactivation reactionshowed a time course consisting of two steps of the first-orderreaction. The inactivated enzyme preparation was not reactivatedby thiol compounds such as cysteine, 2-mercaptoethanol and glutathione,although these reagents, to a certain extent, protected theenzyme from inactivation. The results suggest that denaturationof the enzyme protein was involved in inactivation of the enzyme. ¹Part 74 of the phytopathological chemistry of sweet potatowith black rot and injury. ²Present address: Department of Biology, Faculty of Science,Tokyo Metropolitan University, Setagaya-ku, Tokyo. (Received August 5, 1968; )     

A sulfite-dependent adenosine triphosphatase of Thiobacillus thiooxidans. Its partial purification and some properties

A sulfite-dependent ATPase [EC 3.6.1.3 [EC] ] of Thiobacillus thiooxidanswas activated and solubilized by treatment with trypsin [EC3.4.4.4 [EC] ], and purified 84-fold with a 32% recovery. It requiredboth Mg²⁺ and SO₃^2– for full activity, and its optimumpH was found at 7.5–8.0. Mn²⁺, Co²⁺, and Ca²⁺ could partiallysubstitute for Mg²⁺, while SeO₃^2– and CrO₄^2– couldpartially substitute for SO₃^2–. The enzyme hydrolyzed ATP and deoxy-ATP most rapidly and otherphosphate esters were poorer substrates. The apparent Km valuefor ATP was 0.33 mM. The enzyme activity was strongly inhibitedby 0.2 mM NaN₃ and 10 mM NaF. (Received July 27, 1977; )     

Differences in cold lability of pyruvate, Pi dikinase among C4 species

The cold lability of pyruvate, Pi dikinase in crude leaf extractswas studied in a number of C⁴ plants. The survey included C⁴monocots and dicots and species representingthe three C⁴ subgroups:NADP-malic enzyme, NAD-malic enzyme, and PEP-carboxykinase types. In some species (e.g., Digitaria sanguinalis, Sorghum bicolorand Echinochloa crus-galli), the enzyme was very sensitive tocold treatment (half life of about 8 min at 0?C and 10 to 15min at 10?C). In other species (Panicum miliaceum, Panicum maximumand Panicum texanum), the enzyme was very cold tolerant (retentionof 60 to 85% activity after 60 min at 0?C and 90% activity after60 min at 10?C). Among the plants examined, the most cold sensitivepyruvate,Pi dikinase was found among species of the NADP-malicenzyme subgroup. (Received March 9, 1979; )     

Kinetics of Phenylalanine Ammonia-Lyase and the Effect of L-{alpha}-aminooxy-{beta}-phenylpropionic Acid on Enzyme Activity and Radicle Growth in Germinating Lettuce Seeds

The effects of different concentrations of L--aminooxy-ß-phenyIpropionicacid (AOPP), an analog of L-phenylalanine, on the activity ofphenylalanine ammonia-lyase (PAL, EC 4.3.1.5 [EC] ) and the growthof radicles in 24 h old germinating lettuce (Lactuca salivaL.) seeds were investigated. AOPP causes a significant inhibitionof PAL activity in the seeds (85% inhibition at 10⁴ M). It alsocauses a stimulation of radicle growth at that concentration.The results show that the inhibition of PAL by AOPP may be dueto an irreversible binding of the inhibitor to the enzyme leadingto its inactivation. AOPP also inhibits ethylene biosynthesisin germinating lettuce seeds which could probably explain thestimulation of radicle growth in these seeds. The enzyme shows typical Michaelis-Menten kinetics. The K_m forL-phenylalanine is 4.2 x 10⁵ M. The enzyme does not show anytyrosine ammonia-lyase activity. Various substrate analogs suchas D-phenylalanine, p-fluorophenylalanine, ß-phenyllacticacid, tryptophan and the product of the enzyme reaction, trans-cinnamicacid, inhibit the enzyme competitively. A number of intermediatesand endproducts of the phenylpropanpid pathway, except chlorogenicacid, do not show any inhibition. ¹Scientific contribution number 1423 from the New HampshireAgricultural Experiment Station. (Received May 9, 1986; Accepted September 8, 1986)     

Modulation by Bicarbonate of Catalytic and Regulatory Properties of C4Phosphoenolpyruvate Carboxylase from Amaranthus hypochondriacus: Desensitization to Malate and Glucose 6-Phosphate and Sensitization to Mg2+

The catalytic and regulatory properties of phosphoenolpyruvate(PEP) carboxylase (PEPC) are modulated remarkably by the increasein the level of bicarbonate in the assay medium. The activityof PEPC increased by two-fold as the concentration of bicarbonatewas raised from 0.05 to 10 mM. During this state, there wasonly marginal effect on K_m for PEP, while the affinity of PEPCto Mg²⁺ increased by >2 fold. In contrast, the sensitivityof PEPC to malate decreased with increasing concentration ofHCO₃^–. Similarly, the stimulation by glucose 6-phosphate(G-6-P) at optimal concentration (10 mM) of HCO₃^– wasmuch less than that at suboptimal concentration (0.05 mM). K₁for malate increased by about 3 fold and K_a for G-6-P risedby fourfold as bicarbonate concentration was rised from 0.05to 10 mM. These results suggest that HCO₃^– desensitizesPEPC to both malate and G-6-P. Further, these changes were manifestedin both dark- as well as light-forms of the enzyme. Similarresults were obtained with PEPC in leaf extracts or in purifiedform. We therefore propose that bicarbonate-induced changesare independent of phospho-rylation and possibly through a significantchange in the conformation of the enzyme. This is the firstdetailed report indicating marked modulation of regulatory andcatalytic properties of PEPC by bicarbonate, one of its substrate. (Received April 14, 1998; Accepted September 22, 1998)     

Effects of Calcium on NAD(H)-Glutamate Dehydrogenase from Turnip (Brassica rapa L.) Mitochondria

The effect of Ca²⁺ and ammonia on mitochondrial NADH-glutamatedehydrogenase (GDH: EC 1.4.1.2 [EC] ) isolated from turnip root (Brassicarapa L.) activity was examined. Increasing the ammonia [(NH₄)₂SO₄]concentration led to significant substrate inhibition whichcould be reversed by micromolar levels of Ca²⁺. The sensitivityof the enzyme to ammonia inhibition and its reversal by Ca²⁺was affected by proteolysis. After treatment with various proteases,lower concentrations of Ca²⁺ were capable of fully activatingthe enzyme or overcoming the inhibitory effects of high ammonium,compared to non-treated enzyme. However, the protease-treatedenzyme was still sensitive to ethylene glycol-bis(ß-aminoethylether) N,N,N',N'-tetraacetate (EGTA). In contrast, NADH-GDHactivity was inhibited approx. 30% by organic mercurials (200µm), but the residual activity was not affected by thesubsequent additions of EGTA. NADH-GDH activity could also bestimulated by additions of high concentrations of NaCl (300mM) in the absence of added Ca²⁺. These results suggest thathydrophobic and -SH groups may be involved in the regulationof mitochondrial NADH-GDH activity by Ca²⁺. ² Present address: CSIRO Division of Horticulture, Urrbrae,S.A. 5064, Australia (Received April 18, 1990; Accepted July 23, 1990)     

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)     

Contrasting Sensitivities of Chlorella and Higher Plant Phosphofructokinases to Dilution

A partially purified preparation of phosphofructokinase fromChlorella pyrenoidosa became unstable upon dilution. At pH 7.7and 2°C, enzyme activity remained relatively constant whenthe protein concentration was 12 mg/ml, but at 3 mg/ml almostall activity was lost after 95 min. Greater stability was affordedby lower pH, higher temperature, or the presence of fructose-l,6-P₂,ATP or P₁. Phosphofructokinases from the leaves of spinach,oat and lucerne did not show the instability characteristicsof the Chlorella enzyme. ¹Present address: Department of Biochemistry and Nutrition,University of New England, Armidale, N.S.W. 2351, Australia. (Received December 17, 1980; Accepted November 25, 1980)     

Purification and Immunochemical Characterization of NADP-Dependent Glyceraldehyde 3-Phosphate Dehydrogenase of Euglena gracilis

NADP-dependent glyceraldehyde 3-phosphate dehydrogenase fromEuglena gracilis (EC 1.2.1.13 [EC] ) was purified about 170-fold bya two-step procedure involving DEAE-SH cellulose chromatographyand affinity chromatography on ADP-Sepharose. The homogeneousenzyme from mildly sonicated cells contained equal amounts oftwo types of subunits with mol wts of 34,000 (A) and 38,000(B). The active enzyme had a mol wt 144,000 and is thereforean A₂B₂ tetramer. Enzyme from strongly sonicated Euglena cellscontained, in addition, a second allomer with a probable A₄structure. NADdependent glyceraldehyde 3-phosphate dehydrogenase,a tetramer with 36,000 mol wt subunits, was unrelated immunologicallyto the NADP-dependent enzyme although the latter also showedminor NAD-dependent activity. Both isoenzymes of the NADPlinkedglyceraldehyde 3-phosphate dehydrogenase, however, were immunologicallyidentical. ¹Dedicated, to Prof. Dr. O. H. Volk on his 80th birthday. (Received October 13, 1982; Accepted March 21, 1984)     

Some Properties of the Arginine Decarboxylase in Vicia faba Leaves

Growth of Vicia faba seedlings is accompanied by a rapid increasein arginine decarboxylase (EC 4.1.1.19 [EC] ) in the leaves and epicotyl.Increased enzyme activity was observed under saline conditionsin the presence of NaCl and with osmotic stress by mannitol.The partially purified enzyme (about 86-fold) readily decarboxylatedL-arginine, while D-arginine, L-homoarginine, L-ornithine andL-lysine were decarboxylated very slowly, and L-citrulline andL-glutamic acid were not decarboxylated. The K_m value was 5.8?10^–4M for L-arginine. The optimal pH and temperature for activitywere 8.5 and 45?C, respectively. p-Chloromercuribenzoate andN-ethylmaleimide were effective inhibitors of the enzyme. Inhibitionby spermidine, putrescine and agmatine suggested a possiblefeed-back mechanism in the pathway of polyamine biosynthesis. (Received October 11, 1983; Accepted February 24, 1984)     

Collapse of Peroxide-Scavenging Systems in Apple Flower-Buds Associated with Freezing Injury

Changes in the metabolic activities of peroxide-producing systemsand peroxide-scavenging systems after freezing and thawing inflower buds of the apple, Malus pumila Mill., were studied withspecial reference to freezing injury. In flower buds of the‘McIntosh’ apple that were frozen below lethal temperatures,the activity of NADH-Cyt c reductase (EC 1.6.99.3 [EC] ), one of theenzymes in the electron-transport chains that are related tothe peroxide-producing systems, decreased slightly, while thatof Cyt c oxidase (EC 1.9.3.1 [EC] ) hardly changed. By contrast, theactivities of glucose-6-phosphate dehydrogenase (EC 1.1.1.49 [EC] ),dehydroascorbate reductase (EC 1.8.5.1 [EC] ) and ascorbate peroxidase(EC 1.11.1.11 [EC] ), which are involved in the peroxide-scavengingsystems, decreased to very low levels. The activity of glyceraldehyde-3-phosphatedehydrogenase (EC 1.2.1.12 [EC] ) also decreased markedly. However,little change was observed in the activities of hexokinase (EC2.7.1.1 [EC] ), glucosephosphate isomerase (EC 5.3.1.9 [EC] ), glutathionereductase (EC 1.6.4.2 [EC] ) and glutathione peroxidase (EC 1.11.1.9 [EC] ).Examination of substrates involved in the peroxide-scavengingsystems revealed that the levels of glucose-6-phosphate andfructoses-phosphate decreased to approximately 10^–4 to10^–5 M and 10^–5 M, respectively, and the levelsof GSH decreased to about 10^–5 M or became barely detectable.A decrease in the levels of GSSG also occurred while levelsof ascorbate rose slightly. Similar results were observed withflower buds from ‘Starking Delicious’ and ‘Jonathan’apple trees. These results suggest that the freezing injury to apple flower-budsis closely related to the collapse of the peroxide-scavengingsystems that are coupled with the pentose phosphate cycle. Theresults also suggest that the dysfunction of these peroxide-scavengingsystems is caused by H₂O₂, which may be produced during freezingand thawing. (Received March 14, 1992; Accepted June 5, 1992)     

Stimulatory Effect of Chloride Ions on NADP Malic Enzyme from Leaves of two C4 Species of Cyperus

NADP malic enzyme (EC 1.1.1.40 [EC] ) from leaves of two C₄ speciesof Cyperus (C. rotundus and C. brevifolius var leiolepis) exihibiteda low level of activity in an assay mixture that contained lowconcentrations of Cl^–. This low level of activity wasmarkedly enhanced by increases in the concentration of NaClup to 200 mM. Since the activity of NADP malic enzyme was inhibitedby Na₂SO₄ and stimulated by relatively high concentration ofTris-HCl (50–100 mM, pH 7–8), the activation ofthe enzyme by NaCl appears to be due to Cl^–. Variationsin the concentration of Mg²⁺ affected the K_A (the concentrationof activator giving half-maximal activation) for Cl^–,which decreased from 500 mM to 80 mM with increasing concentrationsof Mg²⁺ from 0.5 mM to 7 mM. The K_m for Mg²⁺ was decreased from7.7 mM to 1.3 mM with increases in the concentration of NaClfrom zero to 200 mM, although the increase of V_max was not remarkable.NADP malic enzyme from Cyperus, being similar to that from otherC₄ species, was able to utilize Mn²⁺. The K_m for Mn²⁺ was 5mM, a value similar to that for Mg²⁺. The addition of 91 mMNaCl markedly decreased the K_m for Mn²⁺ to 20 +M. NADP malicenzyme from Setaria glauca, which contains rather less Cl^–than other C₄ species, was inactivated by concentrations ofNaCl above 20 mM, although slight activation of the enzyme wasobserved at low concentrations of NaCl at pH7.6. (Received February 20, 1989; Accepted June 12, 1989)     

Properties of Phosphoenolpyruvate Carboxylase and Carbohydrate Accumulation in the C3-CAM Intermediate Sedum telephium L. Grown Under Different Light and Watering Regimes

A comparison of the activity and properties of the enzyme phosphoenolpyruvatecarboxylase (PEPC) was made for plants of Sedum telephium L.grown under low (70 µmol m^–2 s^–1) or high(500µmol m^–2 s^–1) PPFD and subjected to varyingdegrees of water stress. Under well-watered conditions onlyplants grown under high PPFD accumulated titratable acidityovernight and the extractable activity of PEPC was almost 2-foldhigher in these plants than in plants grown under low PPFD.Increasing drought stress resulted in a substantial increasein the activity of PEPC extracted both during the light anddark periods and a decrease in the sensitivity to inhibitionby malic acid. The magnitude of these changes was determinedby the severity and duration of drought and by light intensity.A comparison of the kinetic properties of PEPC from severelydroughted plants revealed that plants droughted under high PPFDhad a lower K_m for PEP than plants under low PPFD. Additionof 2·0 mol m^–3 malate resulted in an increase inthe K_m for PEP, with plants draughted under low PPFD havinga significantly higher K_m in the presence of malic acid comparedto those under high _PPFD. Response to the activator glc-6-P,which lowered the K_m for PEP, also varied between plants grownunder the two light regimes. Under well-watered conditions PEPCextracted from plants under high PPFD was more sensitive toactivation by glc-6-P than those under low PPFD. After the severedrought treatment, however, the K_m for PEP in the presence ofglc-6-P was similar for enzyme extracted from plants grown underboth light regimes. Soluble sugars and starch were depletedovernight and were both possible sources of substrate for PEPC.With increasing drought, however, the depletion of starch relativeto soluble sugars increased under both light regimes. The propertiesof PEPC and the characteristics of carbohydrate accumulation/depletionare discussed in relation to the regulation of CAM in S. telephiumgrown under different light and watering regimes. Key words: PEP carboxylase, CAM, carbohydrates, Sedum telephium     

The enzyme system for the entrance of 14CO2 in the dark CO2-fixation of brown algae

High activity of phosphoenolpyruvate (PEP)-carboxykinase, orADP: oxalacetate (OAA) carboxy-lyase activity (a kind of EC4. 1. 1. 32) was discovered in enzyme extracts or partiallypurified preparations obtained from the brown algae, Eiseniabicyclis, Dictyota dichotoma, Spatoglossum pacificum; and Hizikiafusiformis. Enzyme activities were determined by measuring theradioactivity incorporated in the products of dark ¹⁴CO₂-fixationand by spectrophotometric determinations. Except for the lowactivity of "malic enzyme" (EC 1. 1. 1.40), no activities ofother carboxylases, i.e. PEP-carboxylase, PEP-carboxytransphosphorylase,and pyruvate carboxylase could be detected in algal extractsprepared under various conditions. Malate dehydrogenase (EC1. 1. 1. 37), fumarase (EC 4. 2. 1. 2), and glutamic: oxalacetictransaminase (EC 2. 6. 1. 1) were also detected. The algal PEP-carboxykinase required ADP and Mn²⁺ for maximumactivity in the carboxylation reaction; and ATP and Mn²⁺, butnot GTP, for maximum activity in both the decarboxylation andOAA-¹⁴CO₂-exchange reactions. The optimum pH of purified PEP-carboxykinase was in the regionof 7.0 to 7.3 in both the carboxylation and decarboxylationreactions, and its Km values for HCO₃^–, PEP, and ADP were10 mM, 0.3 mM, and 0.07 mM, respectively, in the carboxylationreaction, and values for OAA and ATP were 0.05 mM and 0.4 mM,respectively, in the decarboxylation reaction. Furthermore,the decarboxylation reaction was markedly inhibited by 20 mMHCO₃^–. The physiological role of PEP-carboxykinase as the enzyme responsiblefor the entrance reaction of the dark CO₂-fixation is discussed. ¹ Contributions from the Shimoda Marine Biological Station ofTokyo Kyoiku University, No. 236. This work was supported inpart by a Grant-in-Aid for Co-operative Research from the Ministryof Education, Japan and Matsunaga Science Foundation (to T.Ikawa). ² Present address: Department of Antibiotics, the National Instituteof Health, Shinagawa, Tokyo, Japan. (Received February 22, 1972; )     

Glucose 6-phosphate dehydrogenase from sweet potato: Effect of various ions and their ionic strength on enzyme activity

Activity of glucose 6-phosphate dehydrogenase (D-glucose 6-phosphate:NADP oxidoreductase, EC 1.1.1.49 [EC] ) preparation from sweet potatoroot tissue was markedly altered in the presence of variousions. Cations or anions were effective in the following order:Na^$, K^$>Tris^$>NH₄^$>Mg^2$>Ca^2$, or Cl^–>NO₃^–,HPO₄^2–>SO₄^2–>HCO₃^–. Activity was inhibitedat high concentrations of Ca^2$, and HCO₃^–,. In an investigationon the dependence of the activity on pH, two activity peakswere clearly observed at low ionic strength. Ionic strength altered both the Km and Vmax for glucose 6-phosphate(G6P). A Lineweaver-Burk plot for the enzyme, with respect toG6P, showed a bimodal nature at low ionic strength; suggestingnegative cooperativity. Deviation from linearity of the plotwas less with an increase in the ionic strength. ¹ Present address: Institute of Applied Microbiology, Universityof Tokyo, Bunkyo-ku, Tokyo 113. (Received September 18, 1971; )     

Purification, Properties and Phosphorylation of Anaerobically Induced Enolase in Echinochloa phyllopogon and E. crus-pavonis

Enolase (2-phospho-D-glycerate hydrolyase, EC 4.2.1.11 [EC] ) activityis differentially induced by anoxia in the flood-tolerant speciesE. phyllopogon (Stev.) Koss and the flood-intolerant speciesE. crus-pavonis (H.B.K.) Schult. To examine the regulation ofenolase at the protein level, we purified the enzyme from bothspecies to near homogeneity and compared their physico-chemicaland catalytic properties. Enolase purified from E. phyllopogonexhibits optimal activity at pH 7.0, a K_m of 80 µM for2-PGA, a Q₁₀ of 1.97 and an E_a of 12.3 kcal mol^-1. Similarly,enolase from E. crus-pavonis exhibits optimal activity at pH7.0, a K_m of 50 µM for 2-PGA, a Q₁₀ of 2.04 and an E_aof 12.9 kcal mol^-1. The enzyme from both species is thermostable(100% active after 15 min, 50°C) and is a homodimer of 52.5kDa subunits as resolved by SDS-PAGE and immunoblotting. E.phyllopogon enolase was phosphorylated in vitro using either[