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)     

Lamprothamnium, a Euryhaline Charophyte: I. OSMOTIC RELATIONS AND MEMBRANE POTENTIAL AT STEADY STATE

The charophyte Lamprothamnium papulosum (Wallr.) J. Gr. is foundat salinities varying from nearly fresh water to twice thatof sea water. It can maintain its turgor constant at 302 mosmolkg^–1 (0.73 MPa) when exposed to external osmotic pressuresof 550 to 1350 mosmol kg^–1 (1.3–3.3 MPa). Turgorshows a tendency to rise slightly at lower osmotic pressure(388 mosmol kg^–1 of turgor at 150 mosmol kg^–1 externalosmolality). K⁺ and Cl^– are the main solutes in the vacuole,and are most important in controlling internal osmotic pressure.Mg²⁺, Ca²⁺, and SO^2–₄ are present in significant amountsbut their concentrations do not change with changes in externalsalinity. Na⁺ is present in lower concentration than K⁺, andplays a minor role in regulating turgor. Sucrose is presentin significant concentrations, but changes little with changesin salinity. Two enzymes involved in sucrose metabolism, sucrosephosphate synthetase (EC 2.4.1.14 [EC] ), and sucrose synthetase (EC2.4.1.13 [EC] ) are active in whole cell extracts of Lamprothamnium.As in the fresh water charophytes, Lamprothamnium membrane potentialmay be depolarized (close to E_K) or hyperpolarized, and presumablyof electrogenic origin. Both types of potential are found atall salinities tested.     

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 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)     

Participation of Inorganic Orthophosphate in Regulation of the Ribulose-1,5-bisphosphate Carboxylase Activity in Response to Changes in the Photosynthetic Source-Sink Balance

Sink-limited conditions, defined as treatment with continuousillumination, cause a reduction in the rate of photosyntheticfixation of CO₂ in single-rooted leaves of soybean (Glycinemax. Merr. cv. Turunoko). We suggested previously that thisreduction is due to a deactivation of ribulose-1,5-bisphosphatecarboxylase (RuBPcase, E.C. 4.1.1.39 [EC] ) that is caused by a decreasein the level of P_i in the leaves [Sawada et al. (1989) PlantCell Physiol. 30: 691, Sawada et al. (1990) Plant Cell Physiol.31: 697]. In the present study, the mechanism of regulationof RuBPcase activity by P_i was examined. The activity of RuBPcasein the sink-limited leaves, exposed for 6 or 7 d to continuousillumination to alter the source/sink balance, was enhancedwith increasing concentrations of P_i, in a CO₂-free preincubationmedium in the presence of 5 mM MgCl₂ The maximum value [6.3µmole CO₂ (mg Chl)^–1 min^–1] was obtained atapproximately 5 mM P_i after a 5 min incubation, being 3 timesof the activity without the preincubation. The activity of acrude preparation of RuBPcase that had been deactivated by removalof CO₂ and Mg²⁺ ions by the gel filtration was 5.2–9.3nmole CO₂ (mg protein)^–1 min^–1 and was also enhancedby P_i plus Mg²⁺ ions. The maximum value [147–151 nmoleCO₂ (mg protein)^–1 min^–1] was attained at 5 mM P_iafter a 5 min incubation. The cycle of activation and inactivationof deactivated crude RuBPcase was perfectly reversible by additionof P_i to the enzyme and removal of P_i from the enzyme. Levelsof free P_i and of esterified phosphate in the sink-limited leaveswere 69% and 31% of the total phosphate, respectively. By contrast,in the control leaves, these values were 87% and 13%, respectively.These results support our previously stated hypothesis and indicatean important role for free P_i in the regulation of RuBPcaseactivity, in particular in sink-limited plants. (Received February 21, 1992; Accepted July 23, 1992)     

Properties of glutamate dehydrogenase purified from green tobacco callus tissue

Glutamate dehydrogenase [L-glutamate : NAD(P) oxidoreductase(deaminating) EC 1.4.1.3 [EC] .] has been purified from the mitochondrialfraction of green tobacco callus tissue. The enzyme was stableat –20?C for several months. The pH optimum for the aminationreaction was 7.8. But the optimum for the deamination reactionwas indistinct because it was in an extremely alkaline domain.Relative activities of the enzyme for amination were 50 withNADH and 10 with NADPH, and those for deamination were 5 withNAD and 1 with NADP at pH 7.9. The enzyme was inactivated by EDTA, but its activity partiallyrestored by the addition of divalent cations such as Ca²⁺, Mn²⁺,Zn²⁺, Cu²⁺ and Mg²⁺. Ca²⁺, Mn²⁺ and Zn²⁺ activated the reductiveamination 141, 122 and 39% respectively, but these divalentcations scarcely affected the oxidative deamination. Citrate and fumarate acted as inhibitors for reductive amination,and oxaloacetate for oxidative deamination of the enzyme reaction.These inhibitions were counteracted by the addition of Ca²⁺.ATP and ADP exerted an inhibitory effect on both directionsof the enzyme reaction. The inhibitory effect was hardly preventedby the addition of AMP. Ca²⁺ caused considerable recovery fromthe inhibition of ATP and ADP. Amino acids scarcely affectedthe enzyme activity. Michaelis constants were 0.28 mM for NAD, 0.065 mM for NADH,2.19 mM for a-ketoglutarate, 43.6 mM for ammonium chloride and4.24 mM for L-glutamate. ¹To whom requests for reprints should be addressed. (Received June 25, 1980; )     

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)     

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; )     

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; )     

Ion Content of the Halotolerant Alga Dunaliella salina

The intracellular concentration of the major ions in Dunaliellasalina cells were determined, following the removal of extracellularions by ion-exchange minicolumns. Log phase cells, grown inmedia containing 1–4 molar NaCl, contained 30–50mM chloride and 200–350 mM magnesium (5 mM in medium).Phosphorus, which is present intracellularly mostly as polyphosphate,was present in amounts of 60–100 fmoles per cell, equivalentto a concentration of 600–1,000 mM (0.2 mM in medium).Previous data indicated that such cells contained 20–40mM Na⁺, 150–300mM K⁺, 20mM SO^2–₄, and very low concentrationsof Ca2⁺ and charged nitrogenous compounds. Mg²⁺ and K⁺ seemto serve as the major counter ions for the intracellular negativecharge present in the massively accumulated polyphosphates.The former accounts for about 2/3 of the required positive charge.This is supported by the observation that limitation in thephosphate or K⁺ supply in the medium lead to a parallel decreasein the accumulation of intracellular phosphorus, Mg²⁺ or K⁺. ¹Present address: Department of Vegetables, The Volcani Center,Bet-Dagan 50250, Israel. (Received June 13, 1988; Accepted August 25, 1988)     

ATP Synthesis in Cell-Free Extracts of Peanut (Arachis hypogaea L.) Seeds

Cell-free extracts of peanut (Arachis hypogaea L., cv. Shulamit)seeds, incubated with various substrates, synthesized ATP. Significantsynthesis occurred in the presence of AMP + PEP, NADH₂ + PEPand NAD + PEP. When the activities were examined in extractsprepared with 0.3 M mannitol, the rates were 0.6, 0.1 and 0.04nmol min^–1 mg^–1 protein, respectively. The activitiesunder such conditions were linear with time up to 90 min incubationat 30 °C. In the presence of PEP + NADH₂ there was a higherspecific activity in extracts from non-dormant seeds than fromdormant seeds. No such difference was found when PEP + AMP orNAD + PEP was used as the substrate. The temperature dependenceof the activity showed a relatively high energy of activation(Ea) for AMP + PEP and a low one if NADH₂ + PEP or NAD + PEPwas used as substrate. In buffer extracts of seeds ATP was synthesizedin the presence of the above-mentioned substrate combinationsbut the rate of activity exhibited a lag phase at the earlytime of incubation, after which higher rates of activities (ascompared with mannitol extracts) were obtained. The activitieswere Co⁺-dependent, with a K_m of about 0.7 mM. In the bufferextracts relatively high activities of adenylate kinase (EC2.7.4.3 [EC] (AK) and pyruvate kinase (EC 2.7.1.50 [EC] ) (PK) were found.AK was stimulated by ethephon (ethylene). This effect is temperature-dependentand occurs in both directions: in the presence of ADP (ATP +AMP) as well as if ATP + AMP is used as substrate to synthesizeADP. PK is Co⁺-dependent, and unaffected by ethephon. Both activitieswere stimulated by malonate. Key words: Adenylate Kinase, Arachis hypogaea, ATP synthesis, Peanut, Pyruvate kinase, Seed     

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)     

Effect of Cytoplasmic Ca2+ on the Membrane Potential and Membrane Resistance of Chara Plasmalemma

Effects of cytoplasmic Ca²⁺ on the electrical properties ofthe plasma membrane were investigated in tonoplast-free cellsof Chara australis that had been internally perfused with media,containing either 1 mM ATP to fuel the electrogenic pump orhexokinase and glucose to deplete the ATP and stop the pump. In the presence of ATP, cytoplasmic Ca²⁺ up to 2.5?10^–5M did not affect the membrane potential (about -190 mV), butmembrane resistance decreased uniformly with increasing [Ca²⁺]_i.In the absence of ATP, the membrane potential, which was onlyabout -110 mV, was depolarized further by raising [Ca²⁺]_i from1.4?10^–6 to 2.5?10^–5 M. Membrane resistance, whichwas nearly the twofold that of ATP-provided cells, decreasedmarkedly with an increase in [Ca²⁺]_i from zero to 1.38?10^–6M, but showed no change for further increases. Internodal cellsof Nitellopsis obtusa were more sensitive to intracellular Ca²⁺with respect to membrane potential than were those of Charaaustralis, reconfirming the results obtained by Mimura and Tazawa(1983). The effect of cytoplasmic Ca²⁺ on the ATP-dependent H⁺ effluxwas measured. No marked difference in H⁺ effluxes was detectedbetween zero and 2.5?10^–5 M [Ca²⁺]_i; but, at 10^–4M the ATP-dependent H⁺ efflux was almost zero. Ca²⁺ efflux experimentswere done to investigate dependencies on [Ca²⁺]_i and [ATP]_i.The efflux was about 1 pmol cm^–2 s^–1 at all [Ca²⁺]_iconcentrations tested (1.38?10^–6, 2.5?10^–5, 10^–4M).This value is much higher than the influx reported by Hayamaet al. (1979), and this efflux was independent of [ATP]_i. Thepossibility of a Ca²⁺-extruding pump is discussed. ¹ Present address: Botanisches Institut der Universit?t Bonn,Venusbergweg 22, 5300 Bonn, F.R.G. (Received September 22, 1984; Accepted February 19, 1985)     

Influence of SO2 and NO2 Exposure on Glutathione, Superoxide Dismutase and Glutathione Reductase Activities in Scots Pine Needles

A 50-year-old Pinus sylvestris L. stand was exposed for 2 yearsto low concentrations of SO₂ and NO₂ in an open-air exposureexperiment in northern Sweden. The mean SO₂ concentrations inthe centre of the exposed plot during the 1988 exposure from14 June to 25 September, and during the 1989 exposure from 6June to 30 September were 15 nl 1^–1 and 12 nl 1^–1,respectively. The corresponding values for NO₂ were 15 nl 1^–1and 10 nl 1^–1, respectively. The concentration in thecontrol plot was never higher than a few ppb, and mostly below1 nl 1^–1. Needles sampled from the SO₂ and NO₂-exposed area showed reducedactivities of glutathione reductase (GR; EC 1.6.4.2 [EC] ) and superoxidedismutase (SOD; EC 1.15.1.1 [EC] ) compared with controls. The GRactivity showed decreased levels in autumn and winter, whilethe exposure had ceased, and SOD showed decreased activity duringthe second summer of exposure. Neither membrane-bound nor water-solubleanti-oxidants such as -tocopherol, carotenoids or glutathionechanged due to the exposure. The sulphur/nitrogen ratio wasincreased in needles that were exposed to SO₂ and NO₂ implyinga changed nutrition balance. The results suggest that the capacityof SOD and GR in the ascorbate-glutathione pathway was reduceddue to the exposure to air pollutants. Key words: Anti-oxidants, -tocopherol, glutathione, pigments, Pinus sylvestris L     

Effects of ATP, Mg2+, and redox agents on the Ca2+ dependence of RyR channels from rat brain cortex

Despite their relevance for neuronal Ca²⁺-induced Ca²⁺ release (CICR), activation by Ca²⁺ of ryanodine receptor (RyR) channels of brain endoplasmic reticulum at the [ATP], [Mg²⁺], and redox conditions present in neurons has not been reported. Here, we studied the effects of varying cis-(cytoplasmic) free ATP concentration ([ATP]), [Mg²⁺], and RyR redox state on the Ca²⁺ dependence of endoplasmic reticulum RyR channels from rat brain cortex. At pCa 4.9 and 0.5 mM adenylylimidodiphosphate (AMP-PNP), increasing free [Mg²⁺] up to 1 mM inhibited vesicular [³H]ryanodine binding; incubation with thimerosal or dithiothreitol decreased or enhanced Mg²⁺ inhibition, respectively. Single RyR channels incorporated into lipid bilayers displayed three different Ca²⁺ dependencies, defined by low, moderate, or high maximal fractional open time (P_o), that depend on RyR redox state, as we have previously reported. In all cases, cis-ATP addition (3 mM) decreased threshold [Ca²⁺] for activation, increased maximal P_o, and shifted channel inhibition to higher [Ca²⁺]. Conversely, at pCa 4.5 and 3 mM ATP, increasing cis-[Mg²⁺] up to 1 mM inhibited low activity channels more than moderate activity channels but barely modified high activity channels. Addition of 0.5 mM free [ATP] plus 0.8 mM free [Mg²⁺] induced a right shift in Ca²⁺ dependence for all channels so that [Ca²⁺] <30 µM activated only high activity channels. These results strongly suggest that channel redox state determines RyR activation by Ca²⁺ at physiological [ATP] and [Mg²⁺]. If RyR behave similarly in living neurons, cellular redox state should affect RyR-mediated CICR. Ca²⁺-induced Ca²⁺ release; Ca²⁺ release channels; endoplasmic reticulum; thimerosal; 2,4-dithiothreitol; ryanodine receptor     

Estimation of Cytoplasmic Free Mg2+ Levels and Phosphorylation Potentials in Mung Bean Root Tips by In Vivo 31P NMR Spectroscopy

The cytoplasmic [MgATP]/[ATP]_free ratios, free Mg²⁺ concentrations,and phosphorylation potentials in mung bean [Vigna mungo (L.)Hepper] root tip cells were investigated by ³¹P nuclear magneticresonance spectroscopy. ³¹P NMR spectra show well defined peaksdue to G6P, cytoplasmic P_i, vacuolar P_i, ATP, UDP-glucose andnicotinamide adenine nucleotides. The concentrations of phosphorusmetabolites were determined from quantitative ³¹P NMR spectra.The [MgATP]/[ATP]_free ratio was 9.45. Accordingly, about 90%of the cytoplasmic ATP was complexed to Mg²⁺. Utilizing thedissociation constant (K_d) determined for MgATP, the cytoplasmicfree Mg²⁺ concentration was estimated to be 0.4mM. The NMR-derivedphosphorylation potential, [ATP]/([ADP][P_i]), was 960 M^-1. Thesodium azide treatment decreased the [ATP]/[ADP] ratio and thephosphorylation potential, and increased the [Mg²⁺]^free. Metabolicinhibition may have been enhanced by an increase in [Mg^2+freeand a decrease in the free energy change for ATP hydrolysis,which resulted due to a decrease in the ATP level. ¹Present address: National Food Research Institute, TsukubaCity, Ibaraki 305, Japan. (Received February 8, 1988; Accepted June 1, 1988)     

Ontogenetic Changes in the Chemical Composition of Ricinus communis Grown with NO-3 or NH+4 as N Source

Ricinus communis L. var. Gibsonii was grown in Long Ashton nutrientmedium with either 12mol m^–3 NO^–₃ or 8.0 mol m^–3NH⁺₄ as N source. Two plants from each N treatment were harvestedtwice a week and analysed for C, N, P, S, NO^–₃, SO^2–₄Cl^–K⁺Na⁺, Ca²⁺ Mg²⁺ and ash alkalinity. Statistical analysis of thedata showed that the effect of age and N source was differentfor the chemical variables analysed. Thus [Na⁺] was unaffectedby age or N source, and for both N sources [Mg²⁺] started atthe same level and decreased at the same rate as the plantsmatured. With NH⁺₄ as N source, [SO^2–₄] was higher thanwith NO^–₃, but did not alter with age. The concentrations,in mmol g^–1 dry wt, of C, organic N, K⁺ and Ca²⁺ weredifferent for the two N sources, but the levels of these variablesaltered with age in the same way for both N sources; i.e. therewas no age x N interaction. In the case of P, NO^–₃, Cl^– and COO^–, however,age-related variations were different for the two N sources.It is concluded, inter alia, that [Na⁺] is determined by external[Na⁺] alone, and that K⁺, Ca²⁺ and Cl^– are the inorganicions actively involved in charge balance during ion uptake bythe roots. Key words: Ontogeny, Chemical composition, Plant nutrition     

Biosynthesis of folic acid compounds in plants VI. The occurrence and properties of the dihydrofolate-synthesizing enzyme in pea seedlings

An enzyme, which catalyzes the formation of dihydrofolate fromdihydropteroic acid and L-glutamic acid, was found in pea seedlings.The enzyme was purified approximately 25-fold from the crudeextracts of pea seedlings, and its some properties were investigated.Optimum pH for the enzyme activity was found to be 8.8. Pteroicand tetrahydropteroic acids were not active as substrate. Theenzymatic reaction required as cofactors ATP, divalent (Mg²⁺or Mn²⁺) and univalent (K⁺, NH₄⁺ or Rb⁺) cations. The productwas characterized as dihydrofolic acid by bioautography. MICHAELIS constants for L-glutamic acid, ATP, dihydropteroicacid and Mg²⁺ were 7.0x10^–4, 9.0x10^–5, 3.5x10^–6and 1.2x10^–3 M, respectively. The MICHAELIS constant forMn²⁺ was 3.0x10^–4. The enzyme was inhibited by PCMB orsilver nitrate and, to some extent, by L-aspartic acid. Inhibitionby PCMB was completely reversed by addition of 2-mercaptoethanol.Enzyme activity was distributed widely among plants. The importanceof magnesium and potassium ions for enzyme catalysis is discussed. ¹For the previous paper, Part V, see Reference (30). (Received March 28, 1970; )     

Photosynthetic and Photorespiratory Enzymes in Widely Divergent Plant Species with Special Reference to the Moss Ceratodon purpureus: Properties of Ribulose Bisphosphate Carboxylase/ Oxygenase, Phosphoenolpyruvate Carboxylase and Glycolate Oxidase

Rintamäki, E. and Aro, E.-M. 1985. Photosynthetic and photorespiratoryenzymes in widely divergent plant species with special referenceto the moss Ceratodon purpureus: Properties of ribulose bisphosphatecarboxylase/oxygenase, phosphoenolpyruvate carboxylase and glycolateoxidase.—J. exp. Bot. 36: 1677–1684. Km(CO₂) values and maximal velocities of ribulose bisphosphatecarboxylase/oxygenase (E.C. 4.1.1.39 [EC] ) were determined for sixplant species growing in the wild, consisting of a moss, a fernand four angiosperms. The maximum velocities of the RuBP carboxylasesvaried from 0.13 to 0.;62 µmol CO₂ fixed min^–1 mg^–1soluble protein and the Km(CO₂) values from 15 to 22 mmol m^–3CO₂. The highest Km(CO₂) values found were for the moss, Ceratodonpurpureus, and the grass, Deschampsia flexuosa. These plantsalso had the highest ratios of the activities of RuBP carboxylaseto RuBP oxygenase. Glycolate oxidase (E.C. 1.1.3.1 [EC] ) activitieswere slightly lower in D.flexuosa, but not in C. purpureus,than for typical C₃ species. Phosphoenolpyruvate carboxylase(E.C. 4.1.1.31 [EC] ) was not involved in the photosynthetic carboxylationby these two plants. However, another grass, Phragmites australis,was intermediate in PEP carboxylase activity between C₃ andC₄ plants The properties of RuBP carboxylase/oxygenase are discussedin relation to the activities of PEP carboxylase and glycolateoxidase and to the internal CO₂ concentration. Key words: RuBP carboxylase, oxygenase, Km(CO₂), moss     

Relationship between light-induced potential change and internal ATP concentration in tonoplast-free Chara cells

The effect of the intracellular concentration of ATP ([ATP]₁)on the light-induced potential change (LPC) in tonoplast-freeChara cells was studied. The LPC was hardly affected by loweringthe [ATP]₁ by about 1/10 or by raising it to about 10 timesthe normal cytoplasmic concentration (0.5–1.3 mM). Theinsensitivity of LPC to [ATP]₁ excludes the possibility thatan increase in [ATP]₁ due to photosynthesis may induce the LPC.However, extreme lowering of the [ATP]₁ to about 1–2 µMcompletely inhibited LPC, although photosynthetic O₂ evolutionwas not significantly inhibited. This fact supports the hypothesisthat light stimulates the putative H⁺pump fueled by ATP. Theuncoupling agents DNP and CCCP greatly depolarized the membrane,and inhibited LPC strongly, but they did not decrease [ATP]₁.Photosynthetic O₂ evolution was inhibited to some extent by2 µM CCCP and strongly inhibited by 0.1 mM DNP. Sincethe membrane resistance increased significantly, these chemicalsare believed to act on the membrane as an inhibitor of the electrogenicH⁺ pump not as an H⁺conductor. Introduction of 1 mM ATP intocells treated with uncouplers, to a large extent restored theirability to produce LPC although the membrane potential in darknesswas maintained at a low level. ¹Present address: Niigata College of Pharmacy, 5829 Kamishinei-cho,Niigata 950-21, Japan. ²Present address: Department of Agricultural Chemistry, Collegeof Agriculture, Kyoto University, Kyoto 606, Japan. ³Present address: Department of Botany, Faculty of Science,University of Tokyo, Hongo, Tokyo 113, Japan. (Received March 9, 1979; )