Isolation and characterization of adenosine triphosphatase from Salicornia pacifica var. utahensis

Membrane-bound ATPase associated with plasma membrane and solubleATPase associated with the cytoplasm were prepared from shootsof Salicornia pacifica var. utahensis by sucrose density gradientcentrifugation. The isolated ATPases were tolerant to high concentrationsof NaCl. The Km for membrane-bound ATPase was 1.75 mM and forsoluble ATPase, it was 1.4 mM. The relative effectiveness ofdivalent cations for stimulation of membrane-bound ATPase wasMg²⁺>Fe²⁺>Mn²⁺>Co²⁺>Cu²⁺. Soluble ATPase activitywas stimulated by Ba²⁺>Ca²⁺>Mg²⁺ and was inhibited byCu²⁺, Zn²⁺, Co²⁺ and Fe²⁺. The compounds N,N¹-dicyclohexylcarbodiimide,NaF and ADP, did inhibit the ATPases but ouabain, triphenyltinhydroxide, sodium azide, indoleacetic acid and abscisic aciddid not inhibit the ATPases from s. pacifica var. utahensis. ¹ Present address: Department of Biology, Kong-Ju National College,Kong-Ju, Korea. (Received April 1, 1980; )     

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; )     

NAD Kinase in Corn: Regulation by Far Red Light is Mediated by Ca2+ and Calmodulin

Far red light irradiation of intact corn seedlings (Zea maysL.) has neither an effect on the cellular distribution nor onthe Ca²⁺, calmodulin-dependence of the NAD kinase (EC 2.7.1.23 [EC] ).The enzyme is located in the outer mitochondrial membrane andits activity is totally dependent on the presence of both Ca²⁺and calmodulin, independently of the illumination. In intactmitochondria and the presence of calmodulin the enzyme activityincreases linearly from 100 nM to 1 mM. At 100 µM Ca²⁺halfmaximal activation occurs at about 10 nM calmodulin. After solubilizationand purification by calmodulin-Sepharose chromatography theCa²⁺dependence of the enzyme changes. The activation reachesa plateau at about 100 µM Ca²⁺ and half maximal activationoccurs at about 6 µM Ca²⁺. On the other hand irradiationof intact corn seedlings as well as an increase of the cellularCa²⁺ concentration leads to an increase of NADP and a correspondingdecrease of NAD. Based on these data we suggest that the lighteffect on the NAD kinase activity is mediated by Ca²⁺ and calmodulin. (Received May 31, 1986; Accepted July 14, 1986)     

Alicyclic acid metabolism in plants 12. Partial purification and some properties of shikimate kinase from Phaseolus mungo seedlings

Shikimate kinase from Phaseolus mungo seedlings was partiallypurified by DEAEcellulose, hydroxyapatite and Sephacryl S-200column chromatographies. The activity was completely inhibitedby EDTA and the requirement for Mg²⁺ could be partially replacedby Mn²⁺, Ca²⁺; Co²⁺ and Cd²⁺. Sulfhydryl inhibitor did not inhibitthe enzyme activity. The apparent Km values for shikimic acidand ATP at pH 8.6 were 0.25 mM and 0.38 mM, respectively. Theactivity appeared to be maximal at pH 8.6–9.0. Shikimate-3-phosphateand ADP inhibited the activity slightly. Aromatic amino acids,quinic acid and dehydroquinic acid had no significant effecton the activity. (Received January 11, 1979; )     

An NADP-Glutamate Dehydrogenase from the Green Alga Bryopsis maxima. Purification and Properties

NADP-glutamate dehydrogenase (EC 1.4.1.4 [EC] ; NADP-GDH) was purifiedto electrophoretic homogeneity from the multinuclear-unicellulargreen marine alga in Sipho-nales, Bryopsis maxima, and its propertieswere examined. M_r of the undenatured enzyme was 280 kDa, andthe enzyme is thought to be a hexamer of 46 kDa subunit protein.Optimum pHs for the reductive amination and oxidative deaminationwere 7.5 and 8.2-9.0 respectively. The enzyme displayed NADPH/NADH-specificactivities with a ratio of 18 :1. Apparent K_m values for 2-oxoglutarate,ammonia, NADPH, glutamate and NADP⁺ were 3.0, 2.2, 0.03, 3.2and 0.01 mM respectively. The enzymochemical characteristicsof the GDH were studied and compared to those of other species.The B. maxima GDH was insensitive to 5 mM Ca²⁺ and to 1 mM EDTAin contrast to higher plant NAD-GDHs. Chemical modificationswith DTNB and pCMBS suggested that cysteine residues are essentialfor the enzymatic activity as in other species GDHs. The GDHwas not affected by 1 mM purine nucleotides, suggesting thatthe enzyme is not allosteric, in contrast to animal NAD(P)-GDHsand fungal NAD-GDHs. (Received August 12, 1996; Accepted January 7, 1997)     

ON THE NATURE OF MOSS OXALIC ACID OXIDASE

Moss oxalic acid oxidase freed from catalase by boiling is stronglyinhibited by the "metal-complexing" compounds such as thiocyanate,azide, diethyldithiocarbamate, and hydrosulfite. Inactivatedby dialysis against thiocyanate or azide, the enzyme can bereactivated to a considerable extent by the addition of ferricsalt, cytochrome-c or hemoglobin, not by other metal ions, suchas Cu²⁺, Zn²⁺ , Mn²⁺, and Fe²⁺. Nitrate, chlorate, monoiodoacetate,and iodide also act as strong inhibitors towards moss oxalicacid oxidase. Some enzyme fractions which were obtained by the sodium sulfateprecipitation method were stimulated by Fe³⁺, but not by cytochrome-cor by other metallic ions. This stimulation was inhibited bythiocyanate, azide and monoiodoacetate. ¹ Present address: Biological Institute, University of Toyama,Toyama     

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)     

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; )     

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; )     

Negative Phototropism of Vaucheria terrestris Regulated by Calcium II. Inhibition by Ca2+-Channel Blockers and Mimesis by A23187

As reported in a previous article [Kataoka (1988a) Plant CellPhysiol. 29: 1323], growing apices of the xanthophycean coenocyticalga, Vaucheria terrestris, bends away from a unilateral bluelight (BL) source, if they are simultaneously irradiated withstrong background BL in a solution containing 1–4 mM Ca²⁺.Since the negative bending is a function of the product of theexternal Ca²⁺ concentration and the fluence rate of backgroundBL, a BL-induced Ca²⁺-influx at the apex was hypothesized tobe the cause of the phototropic inversion. The present reportprovides strong evidence for this hypothesis. Addition of theCa²⁺ channel blockers, La³⁺, verapamil, nifedipine and nitrendipineto media containing 4 mM Ca²⁺ completely inhibited the phototropicinversion. By contrast, 1 µM A23187 [GenBank] (plus 4 mM Ca²⁺) notonly enhanced the phototropic inversion under background BL,but also mimicked the background BL; i.e. it caused negativebending under safe red light. Inhibition of phototropic inversionby La³⁺ is also observed under conditions where the algae areirradiated with unilateral BL for 1 week. A BL-dependent Ca²⁺influx and a consequential elevation of the cytoplasmic Ca²⁺-levelin the apical growth region must be involved in the early stepsof phototropic response. A BL-controlled opening of L-type Ca²⁺channels is also suggested. ¹A part of this study was reported at the 3rd Phycological Congressat Melbourne 1988 (Kataoka 1988b) and XXII Yamada Conferenceon Plant Water Relations and Growth Under Stress at Osaka 1989(Kataoka 1989) ²Dedicated to Prof. em. Dr. Noburo Kamiya on the occasion ofhis 77th birthday (Received May 23, 1990; Accepted July 13, 1990)     

The Divalent Cation Pump and its Role in the Regulation of Malic Enzyme Activity in Purified Mitochondria from Potato Tuber

The control of the activity of the matrix-located malic enzyme(EC 1.1.1.39 [EC] ) by Mn²⁺ was investigated in Percoll-purified mitochondriafrom potato (Solarium tuberosum) tuber. Malic enzyme activitywas tightly controlled by the amount of Mn²⁺ available in thematrix space and could be stimulated by the addition of exogenousMn²⁺. A net uptake of Mn²⁺ into the matrix space of energizedmitochondria was measured. The uptake of Mn²⁺ was mediated bythe active cation pump present in the mitochondria. The activityof this cation pump was shown to be dependent on the membranepotential sustained by the activity of the respiratory chain.The uptake of Mn²⁺ was totally abolished in the presence ofan uncoupler and strongly depressed in the presence of rutheniumred, a specific inhibitor of the Ca²⁺-pump which is presentin animal mitochondria. Thus, the effect of Mn²⁺ on matrix-locatedMn²⁺-dependent malic enzyme was strongly influenced by the presenceof an uncoupler or of ruthenium red. In addition, this effectwas reduced in the presence of Ca²⁺. The possible physiologicalsignificance of the presence of this cation pump is discussedin relation to the presence of a matrix-located, NAD⁺-dependentmalic enzyme in plant mitochondria. (Received November 21, 1988; Accepted March 6, 1989)     

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)     

Calcium-Sensitivity of Cortical Microtubules in the Green Alga Mougeotia

Membrane ghosts were prepared from protoplasts of the greenalga Mougeotia, and the Ca²⁺-sensitivity of microtubules onthe ghosts was examined. Microtubules on the protoplast ghosts were not depolymerizedby 3 min treatment with 1 mM Ca²⁺. As the treatment was prolonged,some depolymerization of microtubules became evident, but evenafter 10 min about 50% of the ghosts showed no depolymerization.Ca²⁺ introduced into intact protoplasts seemed to be ineffectivein depolymerizing microtubules; abundant microtubules were presenton membrane ghosts prepared from protoplasts which had beentreated with 2x10^–5M Ca²⁺-ionophore A23187 [GenBank] plus 1 mM Ca²⁺for 20 or 30 min. Neither 3 min treatment with 0.2% Triton X-100 nor with 1 mMCa²⁺ solution containing 5 min MgSO₄ and 100 mM KCl caused depolymerisationof microtubules on protoplast ghosts. However, when given successively,these treatments caused complete depolymerization of microtubules. These results suggest that Mougeotia microtubules are stableto Ca²⁺ and that the stability is conferred by a microtubule-associatedfactor which can easily be removed by Triton X-100 treatment. (Received July 19, 1985; Accepted October 25, 1985)     

Studies on the phosphomannose isomerase of Amorphophallus konjac C. Koch II. Effect of divalent metal ions on the EDTA-treated enzyme

Konjak phosphomannose isomerase was inactivated in a time-dependentprocess by metal binding agents, and the inactivated enzymewas instantaneously reactivated by adding such metal ions asZn⁺⁺, Co⁺⁺, Fe⁺⁺, Mn⁺⁺ and Cu⁺⁺. However, neither Ca⁺⁺ nor Mg⁺⁺were effective for reactivation. Zn⁺⁺, at a low concentration,brought about complete reactivation of the enzyme at pH 6–7. The EDTA-treated enzyme was more susceptible to heat denaturationwhen compared with the native enzyme, but the addition of variousmetal ions caused the recovery of the thermal stability of theEDTA-treated enzyme. The magnitude of the recovery dependedon the metal ion species and the concentrations. The most effectivemetal ion was Co⁺⁺, which caused the recovery of thermal stabilityto a level higher than that of the native enzyme. Phosphomannoseisomerase was inhibited by pchloromercuribenzoate and HgCl₂;the inhibition by p-chloromercuribenzoate being more pronouncedas incubation progressed. In contrast, the EDTA-treated enzymewas more readily inhibited by the mercurial ion than was thenative enzyme. Zn⁺⁺, when added to the EDTA-treated enzyme,markedly restored its resistance to the mercurial-induced inhibition.The metal-substituted enzyme was also inhibited by EDTA in atime-dependent process. ¹ This paper constitutes part 4 of studies on konjak mannanbiosynthesis. (Received March 3, 1975; )     

Control of Glutamate Dehydrogenase from Green Tobacco Callus Mitochondria by Ca2$ and pH

Glutamate dehydrogenase (GDH) (EC 1.4.1.3 [EC] .) purified from greentobacco callus mitochondria was activated markedly by Ca^2$ inthe amination reaction. This activation was detectable evenat concentrations below 5 µM Ca^2$. Saturation curves for the three substrates of the aminationreaction showed normal Michaelis-Menten kinetics in the presenceof 1 mM of Ca^2$, but pronounced substrate inhibition occurredwithout Ca^2$. The effect of Ca^2$ was chiefly on the maximalvelocity. The saturation curve for NH₄Cl in the presence of Ca^2$ was modulatedby a change in pH. The apparent K_m value for NH₄Cl markedlydecreased whereas that for -ketoglutarate increased slightlywhen the pH was raised from 7.3 to 9.0. In contrast, the K_mfor NADH was little affected by raising the pH. The characteristicof GDH which increases its affinity for NH₄Cl when the pH israised may be compatible with the detoxification of ammonia. ¹ Present address: Mochida Pharmaceutical Co., Ltd. (Received August 24, 1981; Accepted November 28, 1981)     

Properties of glutamate dehydrogenase purified from Bacteroides fragilis

The dual pyridine nucleotide-specific glutamate dehydrogenase [EC 1.4.1.3] was purified 37-fold from Bacteroides fragilis by ammonium sulfate fractionation, DEAE-Sephadex A-25 chromatography twice, and gel filtration on Sephacryl S-300. The enzyme had a molecular weight of approximately 300,000, and polymeric forms (molecular weights of 590,000 and 920,000) were observed in small amounts on polyacrylamide gel disc electrophoresis. The molecular weight of the subunit was 48,000. The isoelectric point of the enzyme was pH 5.1. This glutamate dehydrogenase utilized NAD(P)H and NAD(P)+ as coenzymes and showed maximal activities at pH 8.0 and 7.4 for the amination with NADPH and with NADH, respectively, and at pH 9.5 and 9.0 for the deamination with NADP+ and NAD+, respectively. The amination activity with NADPH was about 5-fold higher than that with NADH. The Lineweaver-Burk plot for ammonia showed two straight lines in the NADPH-dependent reactions. The values of Km for substrates were: 1.7 and 5.1 mM for ammonium chloride, 0.14 mM for 2-oxoglutarate, 0.013 mM for NADPH, 2.4 mM for L-glutamate, and 0.019 mM for NADP+ in NADP-linked reactions, and 4.9 mM for ammonium chloride, 7.1 mM for 2-oxoglutarate, 0.2 mM for NADH, 7.3 mM for L-glutamate, and 3.0 mM for NAD+ in NAD-linked reactions. 2-Oxoglutarate and L-glutamate caused substrate inhibition in the NADPH- and NADP+-dependent reactions, respectively, to some extent. NAD+- and NADH-dependent activities were inhibited by 50% by 0.1 M NaCl. Adenine nucleotides and dicarboxylic acids did not show remarkable effects on the enzyme activities.     

Purification and characterization of {delta}-aminolevulinic acid dehydratase from Chlorella regularis

-Aminolevulinic acid dehydratase (-aminolevulinic acid hydrolyaseEC 4.2.1.24 [EC] ) which catalyzes the formation ofporphobilinogenfrom two molecules of -aminolevulinic acid (ALA) was purifiedfrom Chlorella regularis 737-fold by acetone and ammonium sulfatefractionations, DEAE-cellulose column chromatography, and SephadexG-200 gel filtration. The enzyme had an optimum pH of 8.5 inTris-HCl buffer and required either Mg²⁺ or Mn²⁺ for its maximumactivity. The Km values for Mg²⁺, Mn²⁺ and ALA were 15 µM,10µM, and 0.5 mM, respectively. The enzyme was not activatedby thiol compounds, but was inhibited by p-chloromercuribenzoate.The molecular weight estimated by gel filtration was 316,000and the isoelectric point was 5.25. (Received October 18, 1978; )     

Partial purification and some properties of an L-{alpha}-amino-S-caprolactam-hydrolyzing enzyme from Cryptococcus laurentii

A new L-amlnolactam-hydrolyzing enzyme was partially purifiedfrom cells of Cryptococcus laurentii which can grow on L-aminolactamas a carbon and nitrogen source. The enzyme required a bivalentmetal ion, such as Mn²⁺ or Mg²⁺, and its molecular weight wasroughly estimated to be 1.5?10⁵ Some other properties were alsostudied. ¹ Present address: Department of Biology, Faculty of Science,Osaka City University Sumiyoshi-ku, Osaka 558, Japan. (Received June 20, 1977; )     

Enzymic mechanism of starch breakdown in germinating rice seeds V. Sucrose phosphate synthetase in the scutellum

Some enzymic Properties of a partially purified preparationof sucrose phosphate synthetase (E.C.2.4.1.14) from germinatingrice seed scutella were studied. Examination of the reactionkinetics revealed that the rate of synthesis of sucrose phosphatefollows the Michaelis-Menten equation at an optimum PH of 7.5,having K_m of 25 mM for UDP-glucose, and of 4.9 mM for fructose6-phosphate. UDP inhibited the enzyme reaction competitively;K₁ of 3.3 mM. Fe⁺⁺ and Fe⁺⁺⁺ activated the enzyme reaction about2-fold; K_a, 0.3 mM and 2.0 mM, respectively. Co⁺⁺, Co(NH₃)₆⁺⁺⁺,Mg⁺⁺ and Mn⁺⁺ also activated the enzyme reaction. At high concentrationK⁺ activated the enzyme reaction with the maximum activationof 24% at 400 mM. The molecular weight and S_20,w value of theenzyme were determined as 4.5 ? 10⁵ and 10.4S, respectively. ¹Part IV of this series is Ref. (5). ²California Foundation for Biochemical Research Fellow (1973). (Received December 20, 1973; )     

Alicyclic acid metabolism in plants 10. Partial purification and some properties of 3-dehydroquinate synthase from Phaseolus mungo seedlings

Dehydroquinate synthase from Phaseolus mungo seedlings was purified120-fold by DE-23, hydroxylapatite and Sephadex G-100 columnchromatography. The final preparation was free of dehydroquinatehydro-lyase and NAD(P)H₂ oxidase. The dehydroquinate synthaserequired Co²⁺ and NAD as cofactors. Co²⁺ could be replaced byCu²⁺ at 0.1 mM, but Cu²⁺ at higher levels was inhibitory. Noneof the other metal ions tested activated the enzyme. Some activitywas observed in the absence of added Co²⁺ and this activitywas inhibited by EDTA but not by diethyldithiocarbamate, NaN₃or NaCN. Heavy metal ions, such as Ag⁺ and Hg²⁺, and p-chloromercuribenzoatestrongly inhibited the enzyme activity. Of the pyridine nucleotidestested only NAD was required for the maximum activity of theenzyme. In the absence of NAD, the enzyme retained 30 to 40%of the activity obtained with added NAD. The apparent Km valuefor DAHP at pH 7.4 was about 23 µM. The enzyme activityappeared to be maximum at about pH 8.5. However, the characteristicsof the enzyme were studied at pH 7.4, because of the labilityof the enzyme under alkaline conditions. An Arrhenius plot ofthe enzyme reaction showed a break at about 21?C, and belowthis critical temperature the activation energy increased. (Received March 4, 1977; )