Structural and Kinetic Properties of NADP-Malic Enzyme from the Inducible Crassulacean Acid Metabolism Plant Mesembryanthemum crystallinum L.

NADP-malic enzyme (EC 1.1.1.40 [EC] ), which is involved in Crassulaceanacid metabolism (CAM), was purified to electrophoretic homogeneityfrom the leaves of the inducible CAM plant Mesembryanthemumcrystallinum. The NADP-malic enzyme, which was purified 1,146-fold,has a specific activity of 68.8 µmol (mg protein)^–1min^–1. The molecular weight of the subunits of the enzymewas 64 kDa. The native molecular weight of the enzyme was determinedby gel-filtration to be 390 kDa, indicating that the purifiedNADP-malic enzyme is a hexamer of identical subunits. The optimalpH for activity of the enzyme was around 7.2. Double-reciprocalplots of the enzymatic activity as a function of the concentrationof L-malate yielded straight lines both at pH 7.2 and at pH7.8 and did not reveal any evidence for cooperativity of bindingof L-malate. The K_m value for L-malate was 0.35 mM. Hill plotsof the activity as a function of the concentration of NADP⁺indicated positive cooperativity in the binding of NADP⁺ tothe enzyme with a Hill coefficient (nH) of 2.0. An S_0.5 value(the concentration giving half-maximal activity) of 9.9 µMfor NADP⁺ was obtained. Oxaloacetate inhibited the activityof the NADP-malic enzyme. Effects of succinate and NaHCO₃ onthe activity of NADP-malic enzyme were small. (Received October 30, 1991; Accepted May 1, 1992)     

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[     

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.     

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; )     

Sodium Stimulates Regeneration of Phosphoenolpyruvate in Mesophyll Chloroplasts of Amaranthus tricolor

Mesophyll chloroplasts were isolated from leaves of a Na⁺-requiringNAD-malic enzyme type, dicotyledonous C₄ plant, Amaranthus tricolorL. The chloroplasts converted pyruvate to phosphoenolpyruvateunder illumination, and the conversion was stimulated by Na⁺.This observation may explain the requirement for Na⁺ of someC₄ plants. ² Present address: Institute for Life Science Research, NihonNohyaku Co., Ltd., Kawachi-Nagano, Osaka, 586 Japan     

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)     

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)     

Malate Decarboxylation by Mitochondria of the Inducible Crassulacean Acid Metabolism Plant Mesembryanthemum crystallinum

Mitochondria isolated from leaves of Mesembryanthemum crystallinumoxidized malate by both NAD malic enzyme and NAD malate dehydrogenase.Rates of malate oxidation were higher in mitochondria from plantsgrown at 400 mil NaCl in the rooting medium and performing Crassulaceanacid metabolism (CAM) than in mitochondria from plants grownat 20 mM NaCl and exhibiting C₃-photosynthetic CO₂ fixation.The mitochondria isolated from plants both in the CAM and C₃modes were tightly coupled and gave high respiratory control.At optimum pH for malate oxidation (pH 7.0), pyruvate was themajor product in mitochondria from CAM-M. crystallinum, whereasmitochondria from C₃-M. crystallinum produced predominantlyoxaloacetate. Both the extracted NAD malic enzyme in the presenceof CoA and the oxidation of malate to pyruvate by the mitochondriafrom plants in the CAM mode had a pH optimum around 7.0 withactivity declining markedly above this pH. The activity of NAD-malicenzyme, expressed on a cytochrome c oxidase activity basis,was much higher in mitochondria from the CAM mode than the C₃mode. The results indicate that mitochondria of this speciesare adapted to decarboxylate malate at high rates during CAM. ¹Current address: Lehrstuhl für Botanik II, UniversitätWurzburg, Mittlerer Dallenbergweg 64, 8700 Würzburg, WestGermany. ²Current address: KD 120, Chemical Research Division, OntarioHydro, 800 Kipling Avenue, Toronto, Ontario M8Z5S4, Canada. ³Current address: Department of Botany, Washington State University,Pullman, Washington 99164-4230, U.S.A. (Received March 13, 1986; Accepted September 18, 1986)     

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; )     

The occurrence and properties of methenyltetrahydrofolate cyclohydrolase in plants

Methenyltetrahydrofolate cyclohydrolase (E.C. 3.5.4.9 [EC] ), whichis responsible for the enzymatic conversion of 5,10-methenyl-H₄FAto 10-formyl-H₄FA, has been found in various plant tissues.The enzyme was partially purified from pea seedlings and someof its properties were investigated. It was unstable, but wasstabilized by the addition of 25% glycerol. The enzyme was purifiedabout 60-fold by fractionation with ammonium sulfate and columnchromatography on DEAE-cellulose in the presence of 25% glycerol.Optimum pH for the reaction was 7.7. Michaelis constants for5,10-methenyl-H₄FA in the forward reaction, and for 10-formyl-H₄FAin the reverse reaction were 4x10^–5M and 2x10^–4M,respectively. The apparent equilibrium constant for the reactionwas calculated as 50. Enzyme activity was greatly inhibitedby the reduced forms of folate derivatives. The probable participationof this enzyme in the regulation of folate coenzyme levels inplant tissues has been suggested. ¹ Studies on the enzymatic synthesis and metabolism of folatecoenzymes in plants, VI. (For Part V, see Reference (5) ). Partof this paper was presented at the 22nd annual meeting of theJapan Vitamin Society held at Hiroshima on October 14, 1970. ² Present address: Sizuoka Eiwa Junior College, Ikeda, Shizuoka. (Received September 9, 1972; )     

Purification and Characterization of Phosphoenolpyruvate Carboxylase of Photomixotrophically Cultured Green Tobacco Cells

Phosphoenolpyruvate (PEP) carboxylase (PEPCase, EC 4.1.1.31 [EC] )was purified to apparent electrophoretic homogeneity from photomixotrophicallycultured tobacco cells by ammonium sulfate fractionation, DEAE-Sephacel-,hydroxylapatite-, Phenyl-Sepharose CL-4B-, and Sepharose CL-6B-chromatography,and fast protein liquid chromatography on Mono Q. The purifiedenzyme had a specific activity of 32 units per mg protein, andits purity was determined by denaturing polyacrylamide gel electrophoresis.The native enzyme, with a molecular weight of about 440,000,was a tetramer of four identical subunits and showed maximumactivity at pH 8.5–9.0. Non-denaturing isoelectric focusingshowed a single band at pl 5.4. Substrate-saturation kineticsof the purified enzyme for PEP, bicarbonate, and Mg^2$ were typicalMichaelis-Menten type, with K_m-values of 60, 200, and 80µM,respectively. Most effectors which are known to influence theactivity of C₄- or bacterial PEPCase had only small effectson the activity of the purified enzyme at optimum pH, whilesome inhibitory effects by organic acids (malate, citrate andoxaloacetate) and.an activating effect by glucose-6-phosphatewere observed at a suboptimal pH of 7.5. (Received September 30, 1987; Accepted December 14, 1987)     

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)     

Effect of Carbonic Anhydrase on the Activity of Ribulose Bisphosphate Carboxylase

At concentrations of CO₂ less than saturating, carbonic anhydrase(EC 4.2.1.1 [EC] ) stimulates the carboxylation of ribulose bisphosphatecatalysed by ribulose bisphosphale carboxylase (EC 4.1.1.3 [EC] .9)in vitro. This is not through any beneficial association ofthe two enzymes but is a consequence of the increased rate ofconversion of HCO₃^– ion to CO₂, the substrate for thecarboxylation. Carbonic anhydrase should always be includedin reaction mixtures used to determine the Michaelis constantof ribulose bisphosphate carboxylase for CO₂ where fixationof radioactive CO₂ into phosphoglycerate is the basis of rateestimation. The effect is to decrease the value obtained forthe Michaelis constant.     

Enzymes involved in the last steps of the biosynthesis of mannitol in brown algae

Mannitol-1-phosphate dehydrogenase (EC 1.1.1.17 [EC] ) and mannitol-1-phosphatase(EC number yet unassigned) were detected in the brown algae,Spatoglossum pacificum and Dictyota dichotoma. The enzymes wereextracted from algal fronds and their properties were investigatedusing partially purified preparations. Mannitol-1-phosphatase shows maximum activity at pH 7. The enzymehad a narrow substrate specificity. The Km value for mannitol-1-phosphateis 8.3x10^–4 M (30°C, pH 7.0). The enzyme is activatedby Mg⁺⁺ and Mn⁺⁺and is strongly inhibited by PCMB, Hg⁺⁺and NaF. Mannitol-1-phosphate dehydrogenase showed maximum activitiesat pH values 6.5 and 10.2 in reductive and oxidative reactions,respectively. The dehydrogenase also showed narrow substratespecificity; mannitol-1-phosphate and NAD or fructose-6-phosphateand NADH₂ are utilized, respectively, in oxidative and reductivereactions by the enzyme. Km values for these substrates andthe coenzymes are 2.5x10^–4 M and 7.1x10^–5 M forthe first pair and 2.8x10^–4 M and 1.3x10^–5 M forthe latter pair. This enzyme was strongly inhibited by PCMBand Hg⁺⁺, but was only slightly affected by adenosine phosphates. Possible roles of these enzymes in the biosynthesis of mannitolin brown algae are discussed. ¹ Contributions from the Shimoda Marine Biological Station ofTokyo Kyoiku University, No. 233. This work was supported inpart by a Grant-in-Aid for Co-operative Research from the Ministryof Education, Japan and in part by a grant to one of us (T.Ikawa) from the Matsunaga Science Foundation. ² Present address: Chemical and Physical Laboratory, HoechstJapan Research Laboratory, Minamidai, Kawagoe, Japan. (Received February 22, 1972; )     

Isolation and Purification of Mitochondrial Mn-Superoxide Dismutase from the Gymnosperm Pinus sylvestris L.

Manganese superoxide dismutase (Mn-SOD; EC 1.15.1.1 [EC] ) was purifiedfrom germinating seeds of Scots pine (Pinus sylvestris L.) 3days after the start of imbibition. The purification scheduleincluded (NH₄)₂SO₄ fractionation, anion-exchange and hydrophobic-interactionchromatographies and chromatofocusing. Purified Mn-SOD had anapparent specific activity of 4,130 McCord-Fridovich units (mgprotein)^–1. The molecular mass of the holoenzyme was estimatedto be 91 kDa by size-exclusion chromatography, and a molecularmass of 23 kDa was determined by SDS-PAGE. However, isoelectricfocusing demonstrated that the purified enzyme consisted ofthree similarly migrating isoforms, with isoelectric pointsof approximately 6.5. NH₂-terminal amino acid sequencing ofpurified Mn-SOD revealed no differences among the three isoforms.The comparison of the first 32 NH₂-terminal amino acids withsequences of NH₂-terminal amino acids of Mn-SODs from angiospermsreflected the phylogenetic distances between Scots pine, whichis a gymnosperm, and angiospermic species. Cell fractionationsuggested the mitochondrial localization of Mn-SODs and no evidencefor glyoxysomal localization was found. Mn-SOD activity wasabsent from dry seeds. It was detectable at a considerable levelafter imbibition for 24 h, and it was again absent from 3-week-oldseedlings. (Received February 8, 1994; Accepted May 24, 1994)     

The occurrence and properties of dihydrofolate reductase in pea seedlings

Dihydrofolate reductase (E.C. 1.5.1.3 [EC] ) was found in pea seedlingsand was partially purified by treatments with ammonium sulfate,protamine sulfate and by DEAE-cellulose column chromatography.Some properties of the enzyme were investigated. Optimum pHfor the reaction was 6.5. In the enzyme reaction, FAH₂ and NADPH₂were specifically required. MICHAELIS constants for FAH₂ andNADPH₂ were 4.3x10^–6 M and 4.0x10^–5 M, respectively.Folate antagonists such as aminopterin, methotrexate and pyrimethaminewere potent inhibitors of this enzyme. Enzyme activity was almostcompletely inhibited at a concentration of 10^–7 M of aminopterinand methotrexate and 10^–6 M of pyrimethamine. Growth of germinating pea seeds was inhibited by aminopterin,methotrexate and pyrimethamine, and it recovered significantlywith a tetrahydro-derivative of folate, CF, but not with dihydrofolicor folic acid. These results suggest that growth inhibitionof pea seedlings by these antagonists is due to inhibition ofdihydrofolate reductase in seedlings. ¹Studies on the enzymatic synthesis and metabolism of folatecoenzymes in plants IV. (For the previous paper, Part III, seeReference (21)) . Part of this paper was presented at the AnnualMeeting of the Agricultural Chemical Society of Japan held atTokyo on April 4, 1967 (Received October 8, 1969; )     

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)     

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)     

Properties of Leaf NAD-Malic Enzyme from the Inducible Crassulacean Acid Metabolism Species Mesembryanthemum crystallinum

NAD-malic enzyme (NAD-ME) functions to decarboxylate malatein the light in leaves of certain species displaying Crassulaceanacid metabolism (CAM). The properties of NAD-ME in desaltedextracts from the inducible CAM species, Mesembryanthemum crystallinumwere examined. The shapes of the malate saturation curve andthe activity versus pH curve at 10 mM malate were dependenton the presence of the activator CoA. The malate saturationcurve was sigmoidal in the absence of an activator and hyperbolicin the presence of CoA. The pH optimum with 10mM malate andMn²⁺ as cofactor was as low as 6.5 without an activator, andincreased to 7.2 in the presence of CoA. Fumarate activationwas synergistic with CoA above pH 7.2. The enzyme displayedhysteretic behavior under suboptimal assay conditions. Rapid extraction and desalting of the enzyme (<1.5 mim) followedimmediately by assay did not reveal any difference in the propertiesof the enzyme on a day/night basis. It is proposed that diurnalregulation of the enzyme in vivo is mediated by pH and malatelevel without a change in the oligomeric form of the enzyme.The molecular weight of the enzyme was approximately 350,000at pH 6.5 or 7.8. The enzyme obtained from M. crystallinum inthe C₃ mode was very similar to the CAM enzyme except that itdisplayed a lower V_max. ³ Current address: MSU-DOE Plant Research Lab, Michigan StateUniversity, E. Lansing, Michigan, U.S.A. 48824. (Received October 2, 1984; Accepted December 20, 1984)     

Purification and Properties of a Wall-Bound Endo-1,4-{beta}-Glucanase from Suspension-Cultured Poplar Cells

A wall-bound endo-1,4-ß-glucanase (EC 3.2.1.4 [EC] ) wasobtained from a preparation of the cell walls of suspension-culturedpoplar cells and purified to electrophoretic homogeneity bycation-exchange, hydrophobic, and gel-filtration chromatography.The molecular mass was estimated to be 47 kDa by SDS-PAGE and48 kDa by gel filtration on Superdex 200 pg. The isoelectricpoint (pI) was 5.6. The purified enzyme catalyzed the endo-hydrolysisof carboxymethylcellulose with an optimal pH of 6.5, a K_m of1.2 mg ml^-1, and a V_max of 280 units. The purified enzyme specificallyhydrolyzed the 1,4-ß-glucosyl linkages of carboxymethylcellulose,phospho-swollen cellulose, lichenan, xylan and xyloglucan. Theactivity of the enzyme was strongly stimulated by cysteine-HCl.The N-terminal sequence of the enzyme was similar to that ofan extracellular endo-1,4-ß-glucanase found in suspensioncultures of poplar cells and some homology was recognized toavocado fruit-ripening and bean abscission endo-1,4-ß-glucanases. ¹This work was supported in part by a grant from the Toray ScienceFoundation, Japan, and by a Grant-in-Aid from the Ministry ofEducation, Science and Culture of Japan.