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; )     

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; )     

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; )     

The Transport and Metabolism of Urea in Chara australis: II. UREASE AND THE DISTRIBUTION OF TRANSPORTED UREA

The ureolytic enzyme in Chara was investigated. This enzymewas shown to be a urease with an unusually high affinity forurea(K_m = 158 mmol m^-3). Little inhibition of urease activitywas found when intact Chara cells were exposed to the ureaseinhibitors hydroxyurea, acetohydroxamic acid and N-ethylmaleimide,although there was some inhibition of urea uptake. The distribution of radioactivity amongst the amino acid, organicacid and sugar/neutral fractions, determined by ion-exchangechromatography, was very similar whether the Chara internodeswere exposed to ¹⁴C-urea or to H¹⁴CO₃. This suggests that thefraction of the urea-carbon liberated by the urease as CO₂ andretained by the cell is used in photosynthetic carbon-fixation.During the initial 15 min of ¹⁴C-urea uptake, label appearsin the vacuole only in the form of unmetabolized urea. Afterthis time a variety of labelled compounds appear in the vacuole,presumably reflecting the gradual movement of carbon-fixationproducts from the chloroplasts to the cytoplasm and thence intothe vacuole. Key words: Urea transport, metabolism, Chara, urease     

Growth of Phaseolus vulgaris on Various Nitrogen Sources: The Importance of Urease

Rhizobium-inoculatcd plants of Phaseolus vulgaris L. were grownwith different N-sources (nitrate, ammonium, urea) and differentconcentrations of urea. The distribution of growth between plantparts varied with N-sources. Nitrate and ammonium were moreinhibitory to nodulation than urea, which at 40 mol m^–3N had no effect. Urease activity varied in amount and locationover a range of urea concentrations. At higher concentrations,more urea was transported to and increased urease activity wasfound in the shoot Lower levels of activity in plants relianton N₂-fixation were consistent with a ureide-degradation pathwaynot involving urea. Moderate doses of urea could be assimilatedconcomitantly with N₂-fixation. At higher levels of appliedurea, nodulation and ureide transport to the shoots were reduced,although increased growth could not be maintained at concentrationsof applied urea greater than 6.0 mol m^–3 urea N. Key words: Phaseolus vulgaris, growth, nitrogen source, urease     

Abolition of Heavy Metal Toxicity on Kirchneriella lunaris (Chlorophyta) by Calcium

The green alga Kirchneriella lunaris was incubated with variousheavy metals (Cd²⁺, Co²⁺, Mn²⁺, Ni²⁺) in presence/absence ofcalcium (Ca²⁺). The uptake of heavy metal was affected by Ca²⁺.Growth rate was inhibited by all heavy metals applied. In allCa²⁺-containing cultures Kirchneriella exhibited higher ratesof growth than those containing heavy metal alone. Photosynthesis/respirationratio of K. lunaris cells seems to be the determinant in thiswork. Ca²⁺ variably abolished the effects of the heavy metalsstudied. Maximal positive effect of Ca²⁺ was found with Cd²⁺while with Ni²⁺ it was negligible.Copyright 1995, 1999 AcademicPress Cadmium, cobalt, manganese, nickel, calcium, heavy metals, growth, photosynthesis, Kirchneriella lunaris     

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)     

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     

Studies on the control and properties of ornithine transcarbamylase in germinated spores of Geotrichum candidum

The effects of arginine and urea on the levels of ornithinetranscarbamylase (OTC) were investigated in relation to thephysiological functions of this enzyme in Geotrichum candidum.OTC was repressed in germinated spores to half of its initiallevel when exogenous arginine exceeded 12 mM. The repressionof OTC could not be correlated with intracellular arginine concentration.The addition of urea at the final concentration of 0.035 M increasedthe specific activity of OTC by 5.5 and 2.5 fold as comparedto enzyme levels in arginine-repressed spores and control sporesrespectively. Simultaneous addition of urea and arginine duringgermination prevented either arginine repression or urea inductionof OTC. The enzyme was partially purified from germinated sporesand isolated as a single protein band after disc electrophoresis.Two distinct pH optima for the forward reaction (pH 8.8–9)and backward reaction (pH 7.8) were found. Km values for ornithineand carbamyl phosphate were 5 x 10^–3 M and 6.8 x 10^–4so respectively. The Km for citrulline in the catabolic directionwas 1 x 10^–2 M. Enzymes obtained from cell-free extractsof germinated spores could synthetize ATP from citrulline andADP under physiological conditions by coupling the phosphorolysisof citrulline with carbamate kinase activity. The initial rateof germination was stimulated in the presence of citrullineas the sole nitrogen source, as compared to arginine, glutamineor yeast extract. These observations suggest that citrullinemay be catabolized during germination by means of OTC ratherthan via the energy-consuming urea cycle. (Received June 26, 1971; )     

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)     

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; )     

Only the Complete NADH:Nitrate Reductase Activity is Inhibited by Magnesium, not the Partial Activities

In response to in situ dark modulation, or in vitro ATP preincubationof higher plant nitrate reductase, Mg²⁺ inhibits NADH:nitratereductase activity but not MV:nitrate reductase activity incrude extracts. Also for the purified enzyme the complete NADH:nitratereductase activity is inhibited by Mg²⁺, but not the partialMV:nitrate reductase or Cyt c reductase activities. (Received October 13, 1993; Accepted January 24, 1994)     

Characterization of ATPase Activity Associated with Plasma Membrane from Vigna Hypocotyls

A plasma membrane fraction was isolated from the hypocotylsof cowpea {Vigna unguiculata) by a combination of differentialcentrifugation and sucrose density gradient centrifugation.The ATPase activity of this fraction was dependent on divalentcations (Mn²⁺>Mg²⁺>Co²⁺>Ca²⁺>Fe²⁺>Zn²⁺>Ni²⁺)but was not further stimulated by monovalent cations (K⁺ and/orNa⁺). The pH optimum for the activation of ATPase by Mg²⁺ was7.0. This fraction hydrolyzed ATP or UTP as a substrate andthe ATPase activity obeyed a Michaelis-Menten type of kinetics.The K_m for MgATP ranged from 0.65 to 1.1 mM. The ATPase activitywas inhibited by inhibitors such as N, N'- dicyclohexylcarbodiimide,diethylstilbestrol and triphenyltin chloride, all of which arereported to block proton (H⁺) transport in plant cells, butwas insensitive to those of mitochondrial ATPase such as oligomycinand sodium azide. The ATPase activity was not stimulated bytreatment with ionophores (e.g., carbonyl cyanide p-trifluoromethoxyphenylhydrazone,3,5-di-ter-butyl-4-hydroxybenzilidenemalononitrile and valinomycin⁺KCl)which would be expected to dissipate the electrochemical potentialdifference of H⁺ or the membrane potential difference. The characteristics of the ATPase are compared with those ofplasma membrane ATPases of other plants and its possible rolein H⁺-transport is discussed. ¹ Present address: Institute of Applied Biochemistry, Yagi MemorialPark, Mitake, Gifu 505-01, Japan or Laboratory for Plant EcologicalStudies, Faculty of Science, Kyoto University, Kyoto 606, Japan. (Received April 20, 1984; Accepted August 14, 1984)     

Opposite effects of Ni2+ on Xenopus and rat ENaCs expressed in Xenopus oocytes

Opposite effects of Ni²⁺ on Xenopus and rat ENaCs expressed in Xenopus oocytes. Am J Physiol Cell Physiol 289: C946–C958, 2005. First published June 8, 2005; .—The epithelial Na⁺ channel (ENaC) is modulated by various extracellular factors, including Na⁺, organic or inorganic cations, and serine proteases. To identify the effect of the divalent Ni²⁺ cation on ENaCs, we compared the Na⁺ permeability and amiloride kinetics of Xenopus ENaCs (xENaCs) and rat ENaCs (rENaCs) heterologously expressed in Xenopus oocytes. We found that the channel cloned from the kidney of the clawed toad Xenopus laevis [wild-type (WT) xENaC] was stimulated by external Ni²⁺, whereas the divalent cation inhibited the channel cloned from the rat colon (WT rENaC). The kinetics of amiloride binding were determined using noise analysis of blocker-induced fluctuation in current adapted for the transoocyte voltage-clamp method, and Na⁺ conductance was assessed using the dual electrode voltage-clamp (TEVC) technique. The inhibitory effect of Ni²⁺ on amiloride binding is not species dependent, because Ni²⁺ decreased the affinity (mainly reducing the association rate constant) of the blocker in both species in competition with Na⁺. Importantly, using the TEVC method, we found a prominent difference in channel conductance at hyperpolarizing voltage pulses. In WT xENaCs, the initial ohmic current response was stimulated by Ni²⁺, whereas the secondary voltage-activated current component remained unaffected. In WT rENaCs, only a voltage-dependent block by Ni²⁺ was obtained. To further study the origin of the xENaC stimulation by Ni²⁺, and based on the rationale of the well-known high affinity of Ni²⁺ for histidine residues, we designed -subunit mutants of xENaCs by substituting histidines that were expressed in oocytes, together with WT - and -subunits. Changing His²¹⁵ to Asp in one putative amiloride-binding domain (WYRFHY) in the extracellular loop between Na⁺ channel membrane segments M1 and M2 had no influence on the stimulatory effect of Ni²⁺, and neither did complete deletion of this segment. Next, we mutated His⁴¹⁶ flanked by His⁴¹¹ and Cys⁴¹⁷, a unique site for possible heavy metal ion chelation, and, with this quality, most proximal (100 amino acids upstream of the second putative amiloride binding site at the pore entrance), was found localized at M2. Replacing His⁴¹⁶ with arginine, aspartate, tyrosine, and alanine clearly affected amiloride binding in all cases, as well as Na⁺ conductance, as expressed in the xENaC current-voltage relationship, especially with regard to aspartate and tyrosine. However, similarly to those obtained with the WYRFHY stretch, none of these mutations could either abolish the stimulating effect of Ni²⁺ or reverse it to an inhibitory type. epithelia; divalent cations; amiloride; Na⁺; voltage clamp     

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; )     

In squid nerves intracellular Mg(2+) promotes deactivation of the ATP-upregulated Na(+)/Ca(2+) exchanger

We investigated the role of intracellular Mg²⁺(Mg_i²⁺) on the ATP regulation ofNa⁺/Ca²⁺ exchanger in squid axons and bovineheart. In squid axons and nerve vesicles, the ATP-upregulated exchangerremains activated after removal of cytoplasmic Mg²⁺, evenin the absence of ATP. Rapid and complete deactivation of theATP-stimulated exchange occurs upon readmission ofMg_i²⁺. At constant ATP concentration, the effectof intracellular Mg²⁺ concentration([Mg²⁺]_i) on the ATP regulation of exchangeris biphasic: activation at low [Mg²⁺]_i,followed by deactivation as [Mg²⁺]_i isincreased. No correlation was found between the above results and thelevels of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P₂] measured innerve membrane vesicles. Incorporation ofPtdIns(4,5)P₂ into membrane vesicles activates Na⁺/Ca²⁺ exchange in mammalian heart but not insquid nerve. Moreover, an exogenous phosphatase prevents MgATPactivation in squid nerves but not in mammalian heart. It is concludedthat 1) Mg_i²⁺ is an essentialcofactor for the deactivation part of ATP regulation of the exchangerand 2) the metabolic pathway of ATP upregulation of theNa⁺/Ca²⁺ exchanger is different in mammalianheart and squid nerves.

     

Influence of Ni Supply on Growth and Nitrogen Metabolism ofBrassica napusL. Grown with NH4NO3or Urea as N Source

The significance of nickel (Ni), which is essential for ureaseactivity, for growth and nitrogen (N) metabolism ofBrassicanapusgrown in nutrient solution with either NH₄NO₃or urea assole N source was investigated. Although Ni contents were below25 µg kg^-1d. wt, growth of plants relying on NH₄NO₃wasnot affected by the Ni status. However, supplementing the growthmedium with 0.04 µMNi enhanced dry matter production ofurea-grown plants significantly. Urease activity was significantlyreduced in leaves and roots of plants grown without supplementaryNi irrespective of N source. Plants grown with urea withoutadditional Ni accumulated large amounts of urea and had loweramino acid contents indicating impaired usage of the N supplied,while those grown with NH₄NO₃under Ni-deprived conditions accumulatedendogenous urea in their older leaves. It is suggested thatNi may not be strictly essential for plants receiving mineralN, or that the critical level is well below 25 µg kg^-1d.wt. These results confirm that Ni is required for urease activityand thus for growth of plants on urea-based media, as well asfor recycling endogenous urea.Copyright 1999 Annals of BotanyCompany. Brassica napusvar.annua, amino acids, N nutrition, nickel, spring rape, urea, urease activity.     

Purification and some properties of polyphenol oxidase from root-forming carrot callus tissues

1. Polyphenol oxidase (o-diphenol : O₂ oxidoreductase; E.C.1.10.3.1 [EC] ) was isolated from the other phenolases which werepresent in root-forming carrot callus, and its properties wereexamined. 2. The enzyme was purified about 45-fold over crudeextracts (precipitates between 40–70% saturation widiammonium sulfate) by a combination of Bio-gel filtration, protein-bagfiltration, and carboxymethyl cellulose chromatography. Thepurified oxidase was homogeneous according to polyacrylamidegel electrophoresis and Sephadex gel filtration. It was confirmedby CM-cellulose chromatography that the enzyme was absent incallus tissues without accompanying redifferentiation. 3. Themolecular weight of this oxidase was estimated to be 110,000-120,000 from molecular weight-mobility profiles on polyacrylamidegels containing sodium dodecyl sulfate and molecular size-elutionvolume correlations on Sephadex G-150 columns. 4. The enzymeoxidized o-diphenols but showed no detectable activity againstmonophenols. Pyrocatechol, dopamine, caffeic acid, and chlorogenicacid were effectual substrates of the enzyme with K_m valuesranging from 10^–3 M to 10^–5M. The enzyme effectivelycatalyzed the oxidation of o-diphenols over the range of pH6.0 to 7.0 and was readily inactivated by heating. The enzymeactivity was slightly influenced by increasing ionic strength.The initial rate of the enzymic reaction was enhanced by additionof Cu²⁺, Co²⁺ and Mn²⁺ ions, and was reduced in the presenceof DTT, PCMPS, glycylglycine, and DIECA. (Received June 17, 1978; )     

Purification of Ribulose 5-phosphate Kinase and Minor Polypeptides of Pyrenoid from the Green Alga Bryopsis maxima

Ribulose 5-phosphate (Ru5P) kinase (ATP:D-ribulose 5-phosphate1-phosphotrans- ferase; EC 2.7.1.19 [EC] ), an enzyme in the reductivepentose phosphate cycle, was purified from the green alga Bryopsismaxima and its activity and peptide composition were studied.The specific activity of purified Ru5P kinase was 20 µmoleRuBP formed (mg protein)^–1 min^–1 corresponding toa 490-fold purification from the supernatant of chloroplasts.The K_m values of Ru5P kinase for ATP and Ru5P were 69 µMand 330 µM, respectively. The molecular size of Ru5P kinase was estimated as 90 kDa bygel filtration and that of its polypeptide as 41 kDa by SDS-polyacrylamidegel electrophoresis. A small portion of the Ru5P kinase wasfound in a large molecular state (500 kDa) which was consideredto be an inactive form of the enzyme. Ru5P kinase activity has been reported in the pyrenoid of Eremosphaeraviridis as well as ribulose 1,5-bisphosphate carboxylase-oxygenase(RuBisCO) and ribose 5-phosphate isomerase activity (Holdsworth1971). In Bryopsis maxima, among the pyrenoid polypeptides otherthan that of RuBisCO, we found a polypeptide of 42 kDa, similarto that of Ru5P kinase in molecular size and ratio to RuBisCO.A peptide map of the 42 kDa pyrenoid polypeptide, however, showedthat it differed from that of Ru5P kinase. In conclusion, Ru5Pkinase may be not involved in the pyrenoid of this alga. (Received January 19, 1985; Accepted May 15, 1985)     

Purification and Characterization of a Ca2+-Dependent Protein Kinase from the Halotolerant Green Alga Dunaliella tertiolecta

A Ca²⁺-dependent protein kinase (CDPK) that has been partiallypurified and characterized previously [Yuasa and Muto (1992)Arch. Biochem. Biophys. 296: 175] was further purified to about20,000-fold from the soluble fraction of Dunaliella tertiolecta.The enzyme preparation contained 60- and 52-kDa polypeptidesboth of which phosphorylated casein as a substrate. Both polypeptidesshowed a Ca²⁺-dependent increase in mobility during SDS-PAGEand ⁴⁵Ca²⁺-binding activity after SDS-PAGE and electroblottingonto a nitrocellulose membrane, suggesting that both the 60-and 52-kDa CDPKs directly bind Ca²⁺. The protein kinase inhibitors,K-252a and staurosporine, inhibited the CDPK competitively withrespect to ATP. An antibody raised against the 60-kDa CDPK crossreactedwith both the 60- and 52-kDa polypeptides. Both molecular specieswere autophosphorylated in the presence of Ca²⁺, and a highlyphosphorylated 80-kDa band appeared in addition to these phosphorylatedbands at 60 and 52 kDa in SDS-PAGE. However, the specific activityof CDPK was not changed by prior autophosphorylation when theautophosphorylated enzyme was assayed as a mixture of thesephosphorylated molecular species. Only the 60-kDa polypeptidewas immunodetected in subcellular fractions of Dunaliella cells.The 52-kDa polypeptide increased during storage of the enzyme.These results suggest that the 52-kDa polypeptide is a proteolyticartifact produced during purification. Immunoreactive bandsof 60-kDa were detected in extracts of several green algae butnot in extracts of higher plants or a brown alga. ¹This research was partly supported by Grants-in-Aid from theMinistry of Education, Science and Culture, Japan (No. 06454013and 06304023) and Research Fellowship of the Japan Society forthe Promotion of Science for Young Sciencists. ²Research Fellow (PD) of the Japan Society for the Promotionof Science.