Properties of Phosphoglycolate Phosphatase from Chlamydomonas reinhardtii and Anacystis nidulans

The levels of activity of 2-phosphoglycolate phosphatase in the green algae, Chlamydomonas reinhardtii and Chlorella vulgaris, were in the range of 37 to 60 micromoles per milligram chlorophyll per hour and in the blue-green algae, Anacystis nidulans and Anabaena variabilis were 204 to 310 micromoles per milligram chlorophyll per hour. The activity in each species was similar regardless of whether the algae were grown with air or 5% CO₂ in air. The enzyme purified 530-fold from Chlamydomonas was stable, had a broad pH optimum between 6 and 8.5, and was specific for the hydrolysis of P-glycolate with a K_m of 23 micromolar. The enzyme purified 18-fold from Anacystis was labile, had a sharp pH optimum at 6.3, and was also specific for P-glycolate with a K_m of 94 micromolar. The molecular weight of the enzyme from Chlamydomonas was estimated to be 92,000 by gel filtration.

The phosphatase from both sources required a divalent cation for activity. The Chlamydomonas enzyme was most effectively activated by Co²⁺, but was also activated by Mg²⁺ (K_a = 30 micromolar), Mn²⁺, and Zn²⁺. The Anacystis enzyme was most effectively activated by Mg²⁺ (K_a = 140 micromolar), and was also activated by Co²⁺ and Mn²⁺, but not by Zn²⁺. Anions were also required for maximum activity of the enzyme from both sources. The Chlamydomonas enzyme was activated about 2- to 3-fold by chloride (K_a = 140 micromolar), bromide, nitrate, bicarbonate (K_a = 600 micromolar) and formate. The Anacystis enzyme was activated over 10-fold by chloride (K_a = 870 micromolar), bromide, iodide, and nitrate, but was not activated by bicarbonate or formate.

The properties of the algal enzymes were similar to those previously reported for higher plants. The levels and kinetic properties of the enzyme seemed sufficient to account for the flux through the glycolate pathway that occurs in these algae. The phosphatase was not associated with the ribulose 1,5-bisphosphate carboxylase/oxygenase responsible for P-glycolate formation in the carboxysomes of Anacystis.

     

Characterization of a proton-translocating ATPase in microsomal vesicles from corn roots

Sealed microsomal vesicles were prepared from corn (Zea mays, Crow Single Cross Hybrid WF9-Mo17) roots by centrifugation of a 10,000 to 80,000g microsomal fraction onto a 10% dextran T-70 cushion. The Mg²⁺-ATPase activity of the sealed vesicles was stimulated by Cl⁻ and NH₄⁺ and by ionophores and protonophores such as 2 micromolar gramicidin or 10 micromolar carbonyl cyanide p-trifluoromethoxyphenyl hydrazone (FCCP). The ionophore-stimulated ATPase activity had a broad pH optimum with a maximum at pH 6.5. The ATPase was inhibited by NO₃⁻, was insensitive to K⁺, and was not inhibited by 100 micromolar vanadate or by 1 millimolar azide.

Quenching of quinacrine fluorescence was used to measure ATP-dependent acidification of the intravesicular volume. Quenching required Mg²⁺, was stimulated by Cl⁻, inhibited by NO₃⁻, was insensitive to monovalent cations, was unaffected by 200 micromolar vanadate, and was abolished by 2 micromolar gramicidin or 10 micromolar FCCP. Activity was highly specific for ATP. The ionophore-stimulated ATPase and ATP-dependent fluorescence quench both required a divalent cation (Mg²⁺ ≥ Mn²⁺ > Co²⁺) and were inhibited by high concentrations of Ca²⁺. The similarity of the ionophore-stimulated ATPase and quinacrine quench and the responses of the two to ions suggest that both represent the activity of the same ATP-dependent proton pump. The characteristics of the proton-translocating ATPase differed from those of the mitochondrial F₁F₀-ATPase and from those of the K⁺-stimulated ATPase of corn root plasma membranes, and resembled those of the tonoplast ATPase.

     

Purification of NAD malic enzyme from potato and investigation of some physical and kinetic properties

A procedure is described for purification of NAD malic enzyme (EC 1.1.1.39) to near homogeneity from potato tuber mitochondria. The purified enzyme is active with either NAD or NADP, and functions with either Mg²⁺ or Mn²⁺. V_app is greatest when the enzyme is assayed with Mg²⁺ and NAD. When Mn²⁺ replaces Mg²⁺ the V_app of the NAD-linked reaction decreases but the K_m values for all substrates drop substantially. When NADP is used in place of NAD, the V_app of the Mg²⁺-linked reaction decreases and the K_m values for most substrates increase. The pH optimum of the enzyme depends on the metal ion and cofactor used and varies between 6.4 and 6.8. At pH 6.8, with saturating levels of Mg²⁺ and NAD, the turnover number of the enzyme is 37,000 min^?1. The shape of the pH profile indicates the involvement of two to three protons in the activation of the enzyme, whereas only one proton is involved in the inactivation process. The molecular weight of the enzyme in the presence of 5 mm dithiothreitol and 2 mm MgCl₂ is 490,000 as determined by gel filtration. A lower molecular weight form of the enzyme predominates in gel filtration at lower levels of dithiothreitol and in native gel electrophoresis. Sodium dodecyl sulfate gel electrophoresis of the enzyme reveals two main bands with molecular weights of 61,000 and 58,000, suggesting that the subunit stoichiometry of the high-molecular-weight form may be α₄β₄. However, given the possibility that the smaller subunit may be a proteolytic artifact, the enzyme may prove to be an octamer of identical subunits.     

The influence of the divalent cations Mn2+ and Mg2+ on the activation of particulate and digitonin-solubilized adenylate cyclase from rat fat cell membranes

A comparison was made between the activation of membrane-bound adenylate cyclase from rat fat cell membranes and the enzyme solubilized with digitonin. The isoprenaline stimulation of the particulate enzyme was enhanced by GTP, both in the presence of Mg²⁺ and Mn²⁺, but no effect of the metal ion nor of GTP was found on the K_a of isoprenaline. The K_a of sodium fluoride for enzyme stimulation was shifted to 3-fold higher concentrations when Mg²⁺ was replaced by Mn²⁺, whereas V decreased. GTP did not influence the K_a of sodium fluoride but reduced V, irrespective of the metal ion. After digitonin solubilization the enzyme was no longer responsive to isoprenaline or GTP; however, V of the sodium fluoride activation was higher in the presence of Mn²⁺ than in the presence of Mg²⁺, and the K_a was found at 15-fold higher concentrations. Both the solubilized and the particulate adenylate cyclase were inhibited by adenosine; this inhibition was also seen with the fluoride stimulated enzyme. We conclude that solubilization with digitonin did not result in an enzyme preparation which preferentially turns over MnATP²⁺, although the fat cell adenylate cyclase possesses a metal ion regulatory site with a higher affinity for Mn²⁺ than for Mg²⁺. The data suggest that the guanyl nucleotide regulatory site and the sodium fluoride-sensitive site are located on different subunits while there is an interaction between the metal ion regulatory site and the fluoride-sensitive site.     

Substrate kinetics of the tonoplast h-translocating inorganic pyrophosphatase and its activation by free mg

To clarify the kinetic characteristics and ionic requirements of the tonoplast H⁺-translocating inorganic pyrophosphatase (H⁺-PPiase), PPi hydrolysis and PPi-dependent H⁺ transport were studied in tonoplast vesicles isolated from leaf mesophyll tissue of Kalanchoë daigremontiana Hamet et Perrier de la Bâthie. The tonoplast H⁺-PPiase showed an absolute requirement for a monovalent cation and exhibited hyperbolic kinetics with respect to cation concentration. H⁺-PPiase activity was maximal in the presence of K⁺ (K₅₀ approximately 3 millimolar), with PPi-dependent H⁺ transport being more selective for K⁺ than PPi hydrolysis. When assayed in the presence of 50 millimolar KCl at fixed PPi concentrations, H⁺-PPiase activity showed sigmoidal kinetics with respect to total Mg concentration, reflecting a requirement for a Mg/PPi complex as substrate and free Mg²⁺ for activation. At saturating concentrations of free Mg²⁺, H⁺-PPiase activity exhibited Michaelis-Menten kinetics towards MgPPi²⁻ but not Mg₂PPi, demonstrating that MgPPi²⁻ was the true substrate of the enzyme. The apparent K_m (MgPPi²⁻) for PPi hydrolysis (17 micromolar) was significantly higher than that for PPi-dependent H⁺ transport (7 micromolar). Free Mg²⁺ was shown to be an allosteric activator of the H⁺-PPiase, with Hill coefficients of 2.5 for PPi hydrolysis and 2.7 for PPi-dependent H⁺ transport. Half-maximal H⁺-PPiase activity occurred at a free Mg²⁺ concentration of 1.1 millimolar, which lies within the range of accepted values for cytosolic Mg²⁺. In contrast, cytosolic concentrations of K⁺ and MgPPi²⁻ appear to be saturating for H⁺-PPiase activity. We propose that one function of the H⁺-PPiase may be to act as an ancillary enzyme that maintains the proton-motive force across the vacuolar membrane when the activity of the tonoplast H⁺-ATPase is restricted by substrate availability. As ATP levels decline in the cytosol, free Mg²⁺ would be released from the MgATP²⁻ complex, thereby activating the tonoplast H⁺-PPiase.     

Kinetic properties of NAD malic enzyme from cauliflower

The kinetic characteristics of NAD malic enzyme purified to homogeneity from cauliflower florets have been examined. Free NAD⁺ is the active form of this coenzyme. Double-reciprocal plots of data obtained by varying NAD⁺ and malate^2? at a saturating concentration of Mg²⁺ or by varying Mg²⁺ and NAD⁺ at a saturating level of malate^2? are of intersecting type. This indicates that NAD malic enzyme obeys a sequential mechanism. Analysis of these sets of data suggests that each of these substrate pairs binds randomly to the enzyme. However, each substrate binds tighter when others are already present on the enzyme. NAD malic enzyme cannot decarboxylate malate^2? in the absence of either Mg²⁺ or NAD⁺. Arrhenius plots of the NAD-linked reaction are concave downward, indicating the existence of two rate-determining steps with activation energies of 26.5 and 14.2 kcal/mol, respectively. In addition to Mg²⁺, the enzyme can also use Mn²⁺ and Co²⁺. Using Co²⁺ in place of Mg²⁺ does not change V_max or K_m,malate^2? but the K_m for metal and NAD⁺ are greatly decreased. At pH 7.0 and above, Mn²⁺ isotherms and malate^2? curves with Mn²⁺ are nonlinear and appear to be composed of two separate saturation curves. NAD malic enzyme is completely and irreversibly inactivated by N-ethylmaleimide. The enzyme is also irreversibly inactivated approximately 50% by KCNO.     

Purification and Characterization of Phosphoglycolate Phosphatase from the Cyanobacterium Coccochloris peniocystis

The properties and role of the enzyme phosphoglycolate phosphatase in the cyanobacterium Coccochloris peniocystis have been investigated. Phosphoglycolate phosphatase was purified 92-fold and had a native molecular mass of approximately 56 kilodaltons. The enzyme demonstrated a broad pH optimum of pH 5.0 to 7.5 and showed a relatively low apparent affinity for substrate (K_m = 222 micromolar) when compared to that from higher plants. The enzyme required both an anion and divalent cation for activity. Mn²⁺ and Mg²⁺ were effective divalent cations while Cl⁻ was the most effective anion tested. The enzyme was specific for phosphoglycolate and did not show any activity toward a variety of organic phosphate esters. Growth of the cells on high CO₂ and transfer to air did not result in any significant change in phosphoglycolate phosphatase activity. Competitive inhibition of C. peniocystis triose phosphate isomerase by phosphoglycolate was demonstrated (K_i = 12.9 micromolar). These results indicate the presence of a specific noninducible phosphoglycolate phosphatase whose sole function may be to hydrolyze phosphoglycolate and prevent phosphoglycolate inhibition of triose phosphate isomerase.     

Kinetic and Structural Properties of NADP-Malic Enzyme from Sugarcane Leaves

Oligomeric structure and kinetic properties of NADP-malic enzyme, purified from sugarcane (Saccharam officinarum L.) leaves, were determined at either pH 7.0 and 8.0. Size exclusion chromatography showed the existence of an equilibrium between the dimeric and the tetrameric forms. At pH 7.0 the enzyme was found preferentially as a 125 kilodalton homodimer, whereas the tetramer was the major form found at pH 8.0. Although free forms of l-malate, NADP⁺, and Mg²⁺ were determined as the true substrates and cofactors for the enzyme at the two conditions, the kinetic properties of the malic enzyme were quite different depending on pH. Higher affinity for l-malate (K_m = 58 micromolar), but also inhibition by high substrate (K_i = 4.95 millimolar) were observed at pH 7.0. l-Malate saturation isotherms at pH 8.0 followed hyperbolic kinetics (K_m = 120 micromolar). At both pH conditions, activity response to NADP⁺ exhibited Michaelis-Menten behavior with K_m values of 7.1 and 4.6 micromolar at pH 7.0 and 8.0, respectively. Negative cooperativity detected in the binding of Mg²⁺ suggested the presence of at least two Mg²⁺ - binding sites with different affinity. The K_a values for Mg²⁺ obtained at pH 7.0 (9 and 750 micromolar) were significantly higher than those calculated at pH 8.0 (1 and 84 micromolar). The results suggest that changes in pH and Mg²⁺ levels could be important for the physiological regulation of NADP-malic enzyme.     

Warm growth temperatures decrease soybean cholinephosphotransferase activity

The activity of cytidine 5′-diphosphate (CDP) choline: 1,2-diacylglycerol cholinephosphotransferase (EC 2.7.8.2) in developing soybean (Glycine max L. var Williams 82) seeds was 3 to 5 times higher in cotyledons grown at 20°C than in those grown at 35°C. Some characteristics of the enzyme from cotyledons cultured at 20 and 35°C were compared. In preparations from both growth temperatures, the enzyme showed a pH optimum of 7, K_m of 7.0 micromolar for CDP-choline, and an optimum assay temperature of 45°C. Both enzyme preparations were stimulated by increasing concentrations of Mg²⁺ or Mn²⁺, up to 10 millimolar and 50 micromolar, respectively, though Mn²⁺ produced lower activities than Mg²⁺. Enzymes from both 20 and 35°C show the same specificity for exogenous diacylglycerol. No metabolic effectors were detected by addition of heat treated extracts to the assay mixture. The above findings suggest that the higher enzyme activity at 20°C can be attributed to a higher level of the enzyme rather than to the involvement of isozymes or metabolic effectors. Enzyme activity decreased rapidly during culture at 35°C, indicating a rapid turnover of the enzyme. The level of temperature modulation was found to be a function of seed developmental stage.     

Enzymic Properties of Ribulose-1,5-bisphosphate Carboxylase/Oxygenase Purified from Rice Leaves

The enzymic properties of ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase purified from rice (Oryza sativa L.) leaves were studied. Rice RuBPcarboxylase, activated by preincubation with CO₂ and Mg²⁺ like other higher plant carboxylases, had an activation equilibrium constant (K_cK_Mg) of 1.90 × 10⁵ to 2.41 × 10⁵ micromolar² (pH 8.2 and 25°C). Kinetic parameters of carboxylation and oxygenation catalyzed by the completely activated enzyme were examined at 25°C and the respective optimal pHs. The K_m(CO₂), K_m(RuBP), and V_max values for carboxylation were 8 micromolar, 31 micromolar, and 1.79 units milligram⁻¹, respectively. The K_m(O₂), K_m(RuBP), and V_max values for oxygenation were 370 micromolar, 29 micromolar, and 0.60 units milligram⁻¹, respectively.

Comparison of rice leaf RuBP carboxylase with other C₃ plant carboxylases showed that it had a relatively high affinity for CO₂ but the lowest catalytic turnover number (V_max) among the species examined.

     

UDP-Glucose: (1-->3)-beta-Glucan Synthases from Mung Bean and Cotton: Differential Effects of Ca and Mg on Enzyme Properties and on Macromolecular Structure of the Glucan Product

A re-examination of the kinetic properties of UDP-glucose: (1→3)-β-glucan (callose) synthases from mung bean seedlings (Vigna radiata) and cotton fibers (Gossypium hirsutum) shows that these enzymes have a complex interaction with UDP-glucose and various effectors. Stimulation of activity by micromolar concentrations of Ca²⁺ and millimolar concentrations of β-glucosides or other polyols is highest at low (<100 micromolar) UDP-glucose concentrations. These effectors act both by raising the V_max of the enzyme, and by lowering the apparent K_m for UDP-glucose from >1 millimolar to 0.2 to 0.3 millimolar. Mg²⁺ markedly enhances the affinity of the mung bean enzyme for Ca²⁺ but not for β-glucoside; with saturating Ca²⁺, Mg²⁺ only slightly stimulates further production of glucan. However, the presence of Mg²⁺ during synthesis, or NaBH₄ treatment after synthesis, changes the nature of the product from dispersed, alkali-soluble fibrils to highly aggregated, alkali-insoluble fibrils. Callose synthesized in vitro by the Ca²⁺, β-glucoside-activated cotton fiber enzyme, with or without Mg²⁺, is very similar in size to callose isolated from cotton fibers, but is a linear (1→3)-β-glucan lacking the small amount of branches at C-0-6 found in vivo. We conclude that the high degree of aggregation of the fibrils synthesized with Mg²⁺in vitro is caused either by an alteration of the glucan at the reducing end or, indirectly, by an effect of Mg²⁺ on the conformation of the enzyme. Rate-zonal centrifugation of the solubilized mung bean callose synthase confirms that divalent cations can affect the size or conformation of this enzyme.     

Subcellular Location of NADP-Isocitrate Dehydrogenase in Pisum sativum Leaves

The subcellular location of NADP⁺-isocitrate dehydrogenase was investigated by preparing protoplasts from leaves of pea seedlings. Washed protoplasts were gently lysed and the whole lysate separated on sucrose gradients by a rate-zonal centrifugation. Organelles were located by marker enzymes and chlorophyll analysis. Most of the NADP⁺-isocitrate dehydrogenase was in the soluble fraction. About 10% of the NADP⁺-isocitrate dehydrogenase was present in the chloroplasts as a partially latent enzyme. Less than 1% of the activity was found associated with the peroxisome fraction. NADP⁺-isocitrate dehydrogenase was partially characterized from highly purified chloroplasts isolated from shoot homogenates. The enzyme exhibited apparent K_m values of 11 micromolar (NADP⁺), 35 micromolar (isocitrate), 78 micromolar (Mn²⁺), 0.3 millimolar (Mg²⁺) and showed optimum activity at pH 8 to 8.5 with Mn²⁺ and 8.8 to 9.2 with Mg²⁺. The NADP⁺-isocitrate dehydrogenase activity previously claimed in the peroxisomes by other workers is probably due to isolation procedures and/or nonspecific association. The NADP⁺-isocitrate dehydrogenase activity in the chloroplasts might help supply α-ketoglutarate for glutamate synthase action.     

Glutamine synthetase of pea leaves: divalent cation effects, substrate specificity, and other properties

Purified glutamine synthetase from pea seedlings was most active with Mg²⁺ as the metal activator, but Mn²⁺ and Co²⁺ were 45 to 60% and 30 to 45% as effective, respectively, when assayed at the optimal pH for each cation. The Mg²⁺ saturation curve was quite sigmoid, and evidence indicates that MgATP is the active ATP substance. Co²⁺ also gave a sigmoidal saturation curve, but when Mn²⁺ was varied only slightly sigmoidal kinetics were seen. Addition of Mn²⁺, Ca²⁺, or Zn²⁺ at low concentrations sharply inhibited the Mg²⁺ -dependent activity, partially by shifting the pH optimum. Addition of Co²⁺ did not inhibit Mg²⁺-dependent activity. The nucleotide triphosphate specificity changed markedly when Co²⁺ or Mn²⁺ replaced Mg²⁺. Using the Mg²⁺-dependent assay, the Michaelis constant (Km) for NH₄⁺ was about 1.9 × 10⁻³ M. The Km for l-glutamate was directly proportional to ATP concentration and ranged from 3.5 to 12.4 mm with the ATP levels tested. The Km for MgATP also varied with the l-glutamate concentration, ranging from 0.14 mm to 0.65 mm. Ethylenediaminetetracetic acid activated the enzyme by up to 54%, while sulfhydryl reagents gave slight activation, occasionally up to 34%.     

Differences in the cation sensitivity of adenylate cyclase from heart and skeletal muscle: modification by guanyl nucleotides and isoproterenol

Low concentrations of Mn²⁺ supported the basal adenylate cyclase activity in crude and purified sarcolemmal membranes from cardiac muscle more effectively than did relatively high concentrations of Mg²⁺; at saturating concentrations the cyclase activities obtained with Mg²⁺ or Mn²⁺ were similar. In contrast, Mg²⁺ supported the basal cyclase activities of crude membrane fractions and purified sarcolemmal membranes from skeletal muscle far more effectively than did Mn²⁺; at saturating concentrations of either metal ion the Mg²⁺-supported cyclase activities were 5- to 10-fold greater than Mn²⁺-supported activities. Further, compared to Mg²⁺, Mn²⁺ supported the cyclase activities very poorly in all the primary subcellular fractions of skeletal muscle, whereas this cation was at least as effective as Mg²⁺ in all fractions of cardiac muscle. The apparent affinities of the cyclase for Mn²⁺ in heart as well as skeletal muscle appeared to be greater compared to those for Mg²⁺. The skeletal muscle cyclase displayed greater apparent affinity for MnATP^2? (app. K_m 0.10 mm) compared to MgATP^2? (app. K_m 0.32 mm) whereas the heart enzyme displayed greater apparent affinity for MgATP^2? (app. K_m 0.07 mm) compared to MnATP^2? (app. K_m 0.19 mm). Following preactivation with guanyl-5′-yl imidodiphosphate and isoproterenol, Mn²⁺ (0.15 to 2 mm) supported the cyclase activity of skeletal muscle even more effectively than did optimally effective concentrations of Mg²⁺. With the heart enzyme the relatively greater potency of Mn²⁺ persisted following preactivation. Significant enhancement in the Mn²⁺-sensitivity of skeletal muscle cyclase was also observed when assayed in the presence of GTP and isoproterenol or in the presence of NaF. Preactivation of both heart and skeletal muscle cyclases caused selective enhancement in the enzyme's apparent affinity for free Me²⁺ (Mg²⁺ or Mn²⁺) without influencing the apparent K_m for MeATP^2? (MgATP^2? or MnATP^2?). Evidences were obtained to show that the poor effectiveness of Mn²⁺ in supporting the basal activity of skeletal muscle cyclase is not related to (a) potentiation by Mn²⁺ of adenosine-mediated inhibition of the cyclase, (b) Mn²⁺-induced lability of the cyclase, (c) indirect effects of Mn²⁺ on ATP-regenerating system, or (d) the presence of different cation-specific molecular forms of the cyclase. It is also shown that the onset of enhanced Mn²⁺ sensitivity of the skeletal muscle enzyme following preactivation is not accompanied by a general loss of cation specificity of the cyclase. These results suggest that cations support the catalytic activity of adenylate cyclase by interacting with an enzymeregulatory free metal binding site and that the differential cation sensitivity of nonactivated (basal) cyclases from heart and skeletal muscle is likely due to differences in the properties of such an allosteric metal site. Furthermore, the metal site appears to undergo a conformational change following interaction of the cyclase system with the guanyl nucleotide and isoproterenol since the cation sensitivity of the cyclase and the relative potency of cations depend on the conformational status of the enzyme.     

Plant 5-methylthioribose kinase: properties of the partially purified enzyme from yellow lupin (lupinus luteus L.) seeds

Activity of 5-methylthioribose kinase, the enzyme which catalyzes the ATP-dependent formation of 1-phospho-5-methylthioribose, has been revealed in the extracts from various higher plant species. Almost 2,000-fold-purified enzyme has been obtained from yellow lupin (Lupinus luteus L. cv Topaz) seed extract. Molecular weight of the native enzyme is 70,000 as judged by gel filtration. The lupin 5-methylthioribose kinase exhibits a strict requirement for divalent metal ions. Among the ions tested, only Mg²⁺ and Mn²⁺ acted as cofactors. The curve of kinase initial velocity versus pH reaches plateau at pH 10 to 10.5. The K_m values calculated for 5-methylthioribose and ATP are 4.3 and 8.3 micromolar, respectively.

Among nucleoside triphosphates tested as potential phosphate donors, only dATP could substitute in the reaction for ATP. 5-Isobutylthioribose, an analog of 5-methylthioribose, proved to be the γ-ATP-phosphate acceptor, too. The compound inhibits competitively synthesis of 1-phospho-5-methylthioribose (K_i = 1.4 micromolar). Lupin 5-methylthioribose kinase is completely and irreversibly inhibited by the antisulfhydryl reagent, p-hydroxymercuribenzoate. As in bacteria (Ferro, Barrett, Shapiro 1978 J Biol Chem 253: 6021-6025), the enzyme may be involved in a new, alternative pathway of methionine synthesis in plant tissues.

     

Nicotinamide Adenine Dinucleotide-specific "Malic" Enzyme in Kalanchoë daigremontiana and Other Plants Exhibiting Crassulacean Acid Metabolism

NAD-specific “malic” enzyme (EC 1.1.1.39) has been isolated and purified 1200-fold from leaves of Kalanchoë daigremontiana. Kinetic studies of this enzyme, which is activated 14-fold by CoA, acetyl-CoA, and SO₄²⁻, suggest allosteric properties. Cofactor requirements show an absolute specificity for NAD and for Mn²⁺, which cannot be replaced by NADP or Mg²⁺. For maintaining enzyme activity in crude leaf extracts a thiol reagent, Mn²⁺, and PVP-40 were required. The latter could be omitted from purified preparations. By sucrose density gradient centrifugation NAD-malic enzyme could be localized in mitochondria. A survey of plants with crassulacean acid metabolism revealed the presence of NAD-malic enzyme in all 31 plants tested. Substantial levels of this enzyme (121-186 μmole/hr·mg of Chl) were detected in all members tested of the family Crassulaceae. It is proposed that NAD-malic enzyme in general supplements activity of NADP-malic enzyme present in these plants and may be specifically employed to increase internal concentrations of CO₂ for recycling during cessation of gas exchange in periods of severe drought.     

Characterization of a partially purified adenosine triphosphatase from a corn root plasma membrane fraction

The (K⁺,Mg²⁺)-ATPase was partially purified from a plasma membrane fraction from corn roots (WF9 × Mol7) and stored in liquid N₂ without loss of activity. Specific activity was increased 4-fold over that of the plasma membrane fraction. ATPase activity resembled that of the plasma membrane fraction with certain alterations in cation sensitivity. The enzyme required a divalent cation for activity (Co²⁺ > Mg²⁺ > Mn²⁺ > Zn²⁺ > Ca²⁺) when assayed at 3 millimolar ATP and 3 millimolar divalent cation at pH 6.3. When assayed in the presence of 3 millimolar Mg²⁺, the enzyme was further activated by monovalent cations (K⁺, NH₄⁺, Rb⁺ Na⁺, Cs⁺, Li⁺). The pH optima were 6.5 and 6.3 in the absence and presence of 50 millimolar KCl, respectively. The enzyme showed simple Michaelis-Menten kinetics for the substrate ATP-Mg, with a K_m of 1.3 millimolar in the absence and 0.7 millimolar in the presence of 50 millimolar KCl. Stimulation by K⁺ approached simple Michaelis-Menten kinetics, with a K_m of approximately 4 millimolar KCl. ATPase activity was inhibited by sodium orthovanadate. Half-maximal inhibition was at 150 and 35 micromolar in the absence and presence of 50 millimolar KCl. The enzyme required the substrate ATP. The rate of hydrolysis of other substrates, except UDP, IDP, and GDP, was less than 20% of ATP hydrolysis. Nucleoside diphosphatase activity was less than 30% of ATPase activity, was not inhibited by vanadate, was not stimulated by K⁺, and preferred Mn²⁺ to Mg²⁺. The results demonstrate that the (K⁺,Mg²⁺)-ATPase can be clearly distinguished from nonspecific phosphohydrolase and nucleoside diphosphatase activities of plasma membrane fractions prepared from corn roots.     

Biosynthesis of Cytidine 5'-Diphosphate-diacylglycerol in Endoplasmic Reticulum and Mitochondria of Castor Bean Endosperm

Cytidine 5′-triphosphate (CTP):phosphatidate cytidyltransferase from the endoplasmic reticulum and mitochondria of Ricinus communis L. var Hale was characterized. The endoplasmic reticulum enzyme has a pH optimum of 6.5 and a divalent cation is required, Mn²⁺ being preferred and giving maximum activity at 2.5 millimolar. The estimated K_m for CTP is 16.7 micromolar, but that for phosphatidate could not be determined accurately. The activity was inhibited by both deoxycholate and Triton X-100 at concentrations as low as 0.01% (w/w).

The mitochondrial enzyme has a pH optimum of 6.0 and a divalent cation requirement similar to that of the endoplasmic reticulum. Maximum stimulation of the reaction by substrates occurred with 1.5 millimolar phosphatidate (from egg phosphatidylcholine) and about 400 micromolar CTP. The apparent K_m for phosphatidate could not be estimated accurately since activity was obtained in the absence of added lipid, apparently utilizing endogenous substrate. The K_m estimated for CTP was altered by the presence of the detergent Triton X-100; in its absence the value was 33.3 micromolar, but in its presence the value was 66.7 micromolar. Inclusion of 0.6% (w/w) Triton X-100 in the assay mixture stimulated the activity about 2.5-fold.

     

Purification and Characterization of Cytosolic NADP Specific Isocitrate Dehydrogenase from Pisum sativum

Cytosolic NADP-specific isocitrate dehydrogenase was isolated from leaves of Pisum sativum. The purified enzyme was obtained by ammonium sulfate fractionation, ion exchange, affinity, and gel filtration chromatography. The purification procedure yields greater than 50% of the total enzyme activity originally present in the crude extract. The enzyme has a native molecular weight of 90 kilodaltons and is resolved into two catalytically active bands by isoelectric focusing. Purified NADP-isocitrate dehydrogenase exhibited K_m values of 23 micromolar for dl-isocitrate and 10 micromolar for NADP, and displayed optimum activity at pH 8.5 with both Mg²⁺ and Mn²⁺.     

Isolation and properties of a Mn2+-activated phosphohistone phosphatase from canine heart.

A Mn²⁺-activated phosphohistone phosphatase has been isolated from canine heart. The s_{20, w} for the enzyme is 3.8. Using this value and the value for Stokes radius (39 Å), the molecular weight for the enzyme was calculated to be 61,000. The enzyme is inactive in the absence of divalent cations, among which Mn²⁺ is the most effective activator. Co²⁺ and Mg²⁺ are less effective than is Mn²⁺. Zn²⁺, Fe²⁺, and Cu²⁺ are inhibitory. The enzyme has a pH optimum between 7 and 7.5 and has an apparent K_m for phosphohistone and Mn²⁺ of about 17 μm and 0.5 mm, respectively. The enzyme is inhibited by nucleoside triphosphate, ADP, AMP, phosphate, and pyrophosphate, but is not affected by cyclic AMP or cyclic GMP. The dephosphorylation of phosphohistone is stimulated by salts. Kinetic studies reveal that KCl and other salts greatly affect both the rate of hydrolysis and the K_m for either Mn²⁺ or phosphohistone by interacting with the substrate. The data suggest that modification at substrate level is an important regulatory mechanism for the enzyme. The enzyme preparation also dephosphorylates phosphorylase a and phosphocasein. Evidence suggests that one enzyme possesses both phosphohistone and phosphorylase phosphatase activities and that a different enzyme catalyzes the Mg²⁺- and Mn²⁺-activated dephosphorylation of phosphocasein.