Cyclic AMP-dependent and -independent protein kinases in HeLa cells

Malate saturation isotherms for the NAD⁺ malic enzyme determined at widely differing, but saturating, concentrations (8, 80, 160 mm) of magnesium show the same response to malate concentration only when velocity is plotted against the concentration of free malate^2?. This identification of the ionized malic acid as the true substrate for this enzyme, together with the observation that the complex of Mg with malate has no influence on the reaction rate even at very high concentrations, indicates that the metal ion activator of the enzyme must also bind in the ionized form. A kinetic analysis shows that, with respect to magnesium and malate, the malic enzyme catalyzes a rapid equilibrium reaction of the intersecting type. Either Mg²⁺ or malate^2? may bind first but the fact that the K_m's for both Mg²⁺ and malate^2? are smaller than the respective K_i's suggests that, when either metal ion or malate is present on the enzyme, the other is bound more tightly than when it binds to the free enzyme. This demonstration of the nature of the true substrates for this enzyme has implications for studies of the NAD⁺ malic enzyme in which conditions influencing the amount of free magnesium and malate, e.g., changes in pH, addition of weak acid effectors etc., are involved.     

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.     

Enzymatic characterization of isocitrate dehydrogenase from an emerging zoonotic pathogen Streptococcus suis

Streptococcus suis, a Gram-positive coccus, is an emerging zoonotic pathogen for both humans and pigs, but little is known about the properties of its metabolic enzymes. Isocitrate dehydrogenase (IDH) is a key regulatory enzyme in the citric acid cycle that catalyzes the oxidative decarboxylation of isocitrate yielding α-ketoglutarate and NAD(P)H. Here, we report the overexpression and enzymatic characterization of IDH from S. suis Serotype 2 Chinese highly virulent strain 05ZYH33 (SsIDH). The molecular weight of SsIDH was estimated to be 74 kDa by gel filtration chromatography, suggesting a homodimeric structure. Additionally, SsIDH was divalent cation-dependent and Mg²⁺ was found to be the most effective cation. The optimal pH of SsIDH was 7.0 (Mn²⁺) and 8.5 (Mg²⁺), and the maximum activity was around 30 °C (Mn²⁺) and 50 °C (Mg²⁺), respectively. Heat inactivation studies showed that SsIDH retained 50% activity after 20 min of incubation at 49 °C. Sequence comparison revealed that SsIDH had a significantly homologous identity to bacterial homodimeric IDHs. The recombinant SsIDH displayed a 117-fold (k_cat/K_m) preference for NAD⁺ over NADP⁺ with Mg²⁺, and a 80-fold greater specificity for NAD⁺ than NADP⁺ with Mn²⁺. Therefore, SsIDH has remarkably high coenzyme preference toward NAD⁺. This current work is expected to shed light on the functions of metabolic enzymes in S. suis and provide useful information for SsIDH to be considered as a possible candidate for serological diagnostics and detection of S. suis infection.     

Physical and Kinetic Properties and Regulation of the NAD Malic Enzyme Purified from Leaves of Crassula argentea

The NAD malic enzyme has been purified to near homogeneity from the leaves of Crassula argentea Thunb. The enzyme has two subunits, one of 59,000 daltons, and one of 62,000 daltons. In native gels stained for activity, the enzyme appears to exist in the dimeric, tetrameric, and predominantly the octameric forms.

The enzyme uses either Mg²⁺ or Mn²⁺ as the required divalent cation, and utilizes NADP at a rate less than 20% of that with NAD. With Mn²⁺ the K_m for malate²⁻ is lower than with Mg²⁺, but V_max is lower than with Mg²⁺. In the forward (malate-decarboxylating) direction with NAD, the kinetic parameters are essentially like those observed for the enzyme from C₃ plants. In the reverse reaction, run with Mn²⁺, the activity is 1.5% of that in the forward reaction. The equilibrium constant is 1.1 × 10⁻³ molar.

The kinetic mechanism of the reaction, at least in the forward direction, is sequential, with apparently random binding of all reaction components. Product inhibition patterns confirm this.

The enzyme displays a strong hysteretic lag, which is shortened by high enzyme concentrations, high substrate concentrations, and the presence of the product NADH.

The enzyme is activated by coenzyme A with K_a = 4 micromolar. AMP also shows competitive activation, with K_a = 24 micromolar. The activation by coenzyme A and AMP is additive, implying separate sites for their binding. Phosphoenolpyruvate activates the reaction at low (micromolar) concentrations, but higher concentrations of phosphoenolpyruvate cause deactivation. Fumarate²⁻ is a strong activator, with K_a = 0.3 millimolar. Fructose-1,6-bisphosphate activates the enzyme, but its most pronounced effect is in shortening the lag. Citrate is a competitive inhibitor of malate, with K_i = 4.9 millimolar.

     

Purification and Characterization of NAD Malic Enzyme from Leaves of Eleusine coracana and Panicum dichotomiflorum

NAD malic enzyme (EC 1.1.1.39), which is involved in C₄ photosynthesis, was purified to electrophoretic homogeneity from leaves of Eleusine coracana and to near homogeneity from leaves of Panicum dichotomiflorum. The enzyme from each C₄ species was found to have only one type of subunit by SDS polyacrylamide gel electrophoresis. The M_r of subunits of the enzme from E. coracana and P. dichotommiflorum was 63 and 61 kilodaltons, respectively. The native Mr of the enzyme from each species was determined by gel filtration to be about 500 kilodaltons, indicating that the NAD malic enzyme from C₄ species is an octamer of identical subunits. The purified NAD malic enzyme from each C₄ species showed similar kinetic properties with respect to concentrations of malate and NAD; each had a requirement for Mn²⁺ and activation by fructose- 1,6-bisphosphate (FBP) or CoA. A cooperativity with respect to Mn²⁺ was apparent with both enzymes. The activator (FBP) did not change the Hill value but greatly decreased K_0.5 (the concentration giving half-maximal activity) for Mn²⁺. The enzyme from E. coracana showed a very low level of activity when NADP was used as substrate, but this activity was also stimulated by FBP. Significant differences between the enzymes from E. coracana and P. dichotomiflorum were observed in their responses to the activators and their immunochemical properties. The enzyme from E. coracana was largely dependent on the activators FBP or CoA, regardless of concentration of Mn²⁺. In contrast, the enzyme from P. dichotomiflorum showed significant activity in the absence of the activator, especially at high concentrations of Mn²⁺. Both immunodiffusion and immunoprecipitation, using antiserum raised against the purified NAD malic enzyme from E. coracana, revealed partial antigenic differences between the enzymes from E. coracana and P. dichotomiflorum. The activity of the NAD malic enzyme from Amaranthus edulis, a typical NAD malic enzyme type C₄ dicot, was not inhibited by the antiserum raised against the NAD malic enzyme from E. coracana.     

Reduced nicotinamide-adenine-dinucleotide-pyrophosphorylase: A novel enzyme in seeds

An enzyme which splits reduced NAD has been partially purified from pea (Pisum sativum, Kelvedon Wonder) seeds. The activity requires orthophosphate and the products are ADP and probably NMN (dihydro NMN?). The enzyme splits the NADH₂ at the pyrophosphate bond and incorporates the phosphate into the AMP residue. NAD, NADP or NADPH₂ could not replace NADH₂. The enzyme is unstable during storage, is activated by Mg²⁺ and by Mn²⁺, and inhibited by Ca²⁺. K⁺, Li⁺ and NH₄⁺ have no effect. The possible role of this enzyme in the synthesis of ATP in seeds at the early stage of germination is discussed.     

Active oxidative decarboxylation of malate by mitochondria isolated from L-1210 ascites tumor cells

Mitochondria from L-1210 mouse ascites tumor show a very high rate of oxidation of L-malate in comparison with mitochondria from normal tissues. They were found to contain large amounts of malic enzyme (E.C.1.1.1.39) catalyzing oxidative decarboxylation of L-malate to pyruvate. Malic enzyme in extracts of tumor mitochondria requires Mn²⁺ or Mg²⁺, utilizes either NAD⁺ or NADP⁺ as electron acceptor, and shows positive cooperativity in binding of L-malate. These observations suggest that L-1210 tumor mitochondria actively convert excess tricarboxylate cycle intermediates and their precursors into pyruvate for further oxidation.     

Influence of Certain Cations on Activity of Succinyl CoA Synthetase From Tobacco

Succinyl CoA synthetase from Nicotiana tabacum exhibited a requirement for univalent and divalent cations. Mn²⁺ replaced Mg²⁺ in the assay medium and Co²⁺ and Ca²⁺ partially replaced Mg²⁺. Addition of Zn²⁺ resulted in no enzyme activity. The enzyme was activated by univalent cations K⁺, Rb⁺, NH₄⁺, and Na⁺; Li⁺ showed little or no activation. Maximum enzyme activity varied significantly with potassium salts of different anions. Greatest activation was obtained with K₃PO₄ and, respectively, KCl, KNO₃, K₂SO₄ and KF exhibited steadily decreasing enzyme activation.     

Control of glutamate dehydrogenase from Lemna minor by divalent metal ions

The NADH and NAD⁺ dependent reactions catalyzed by glutamate dehydrogenase (GDH) from sterile cultures of Lemna minor are completely inactivated by EDTA. The activities of both reactions can be fully restored by addition of Ca²⁺ and to a lesser extent Mn²⁺, Zn²⁺, Sr²⁺ or La³⁺, whereas Mg²⁺ reactivates only the NAD⁺ dependent reaction. Activation of the NADH reaction by Ca²⁺ has been studied by using partially purified, EDTA pretreated, and Mg²⁺ saturated GDH preparations. Saturation kinetic curves with Ca²⁺ were always sigmoidal, whereas saturation plots for the 3 substrates of the aminating reaction at various fixed Ca²⁺ concentrations showed normal Michaelis kinetics. However, a pronounced substrate inhibition at low Ca²⁺ levels was found, particularly with NH₄⁺ and NADH. Product inhibition studies revealed unchanged enzyme substrate binding characteristics for NADH and 2-oxoglutarate in the Ca²⁺ free enzyme. A drastic alteration was established for the third substrate NH₄⁺. The kinetic data suggest that Ca²⁺ governs an equilibrium between a catalytically inactive (Ca²⁺ free) and an active (Ca²⁺ saturated) enzyme form. Inactivation by removal of Ca²⁺ is related to an alteration in the binding characteristics or binding sequence of the substrate NH₄⁺.     

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.     

Properties of NADP-malic enzyme from glumes of developing wheat grains

Properties of partially purified NADP-malic enzyme (EC 1.1.1.40) from glumes of developing wheat grains were examined. The pH optimum for enzyme activity was influenced by malate and shifted from 7.3 to 7.6 when the concentration of malate was increased from 2 to 10 mM. The K_m values, at pH 7.3, for various substrates were: malate, 0.76 mM; NADP, 20 μM and Mn²⁺, 0.06 mM. The requirement of Mn²⁺ cation for enzyme activity could be partially replaced by Mg²⁺ or Co²⁺. Mn²⁺ dependent enzyme activity was inhibited by Pb²⁺, Ni²⁺, Hg²⁺, Zn²⁺, Cd²⁺, Al³⁺ and Fe³⁺. During the reaction, substrate molecules (malate and NADP) reacted with enzyme sequentially. Activity of malic enzyme was inhibited by products of the reaction viz pyruvate, HCO₃^? and NADPH₂. At a limiting fixed concentration of NADP, these products induced a positive cooperative response to increasing concentrations of malate.     

Studies on the interaction of metal ions with puruvate kinase from Ehrlich ascites-tumour cells and from rabbit muscle

1. Pyruvate kinase (ATP–pyruvate phosphotransferase, EC 2.7.1.40) from Ehrlich ascites-tumour cells was purified approximately fivefold by chromatography on DEAE-cellulose. The enzyme was shown to have an absolute requirement for one univalent and for one bivalent metal ion. 2. The univalent metal ion requirements were satisfied by K⁺, Rb⁺ or NH₄⁺; Na⁺ and Cs⁺ were weak activators but Li⁺ was inactive. 3. Ca²⁺ exhibited `non-competitive' and `apparent competitive' effects in relation to the K⁺ activation. 4. The bivalent metal ion requirements were satisfied by Mg²⁺, Mn²⁺ or Co²⁺; Ba²⁺, Sr²⁺, Ca²⁺, Ni²⁺, Be²⁺ and Cu²⁺ were inactive. Mn²⁺ and Co²⁺ were better activators than Mg²⁺. 5. The bivalent metal ion requirements of purified pyruvate kinase from rabbit muscle were satisfied by Mg²⁺, Mn²⁺, Co²⁺ and to a smaller extent by Ni²⁺. Mn²⁺ and Co²⁺ were better activators than Mg²⁺. 6. Ca²⁺ competitively inhibited the activation by Mg²⁺, Mn²⁺ and Co²⁺ for both the tumour and rabbit enzymes. 7. It is concluded that there are no significant differences in metal ion specificity between the tumour and rabbit enzymes. 8. The possible role of metal ions in regulating enzymic and metabolic activities is considered further.     

Catalytic properties of d-xylose isomerase from Streptomyces violaceoruber

The catalytic properties and stability of d-xylose isomerase from Streptomyces violaceoruber have been studied. The enzyme was activated by Mg²⁺, Co²⁺ and Mn²⁺ but Ni²⁺, Ca²⁺, Zn²⁺, Cu²⁺ and Hg were ineffective. Optimum catalytic conditions were obtained at 80°C in the pH range 7.5–9.5 and in the presence of 10 mm Mg²⁺. The specific activity of the enzyme increased after treatment with 10 mm EDTA (factor 2.4). A further increase of activity (factor 2.0–2.8) was observed after preincubation of the enzyme with Mg²⁺ or Co²⁺, the preincubation time depending on the incubation temperature. The thermal stability of the enzyme is very high. At 60°C the enzyme retained optimum activity following 30 days of storage in the presence of 1 mm Co²⁺ or 10 mm Mg²⁺. At 80°C, Co²⁺ is superior as a protector against thermal denaturation. At saturating concentrations of Mg²⁺ (35°C) the K_m-values of the EDTA-treated enzyme with respect to d-xylose and d-glucose were 2.8 and 149 mm and the dissociation constants of the enzyme-Mg²⁺ complex for xylitol and d-sorbitol were 0.455 and 4.47 mm, respectively.     

pH Effects on the Activity and Regulation of the NAD Malic Enzyme

The NAD malic enzyme shows a pH optimum of 6.7 when complexed to Mg²⁺ and NAD⁺ but shifts to 7.0 when the catalytically competent enzyme-substrate (E-S) complex forms upon binding malate⁻². This is characteristic of an induced conformational change. The slope of the V_max or V_max/K_m profiles is steeper on the alkaline side of the pH optimum. The K_m for malate increases markedly under alkaline conditions but is not greatly affected by pH values below the optimum. The loss of catalysis on the acidic side is due to protonation of a single residue, pK 5.9, most likely histidine. Photooxidation inactivation with methylene blue showed that a histidine is required for catalytic activity. The location of this residue at or near the active site is revealed by the protection against inactivation offered by malate. Three residues, excluding basic residues such as lysine (which have also been shown to be vital for catalytic activity, must be appropriately ionized for malate decarboxylation to proceed optimally. Two of these residues directly participate in the binding of substrates and are essential for the decarboxylation of malate. A pK of 7.6 was determined for the two residues required by the E-S complex to achieve an active state, this composite value representing both histidine and cysteine suggests that both have decisive roles in the operation of the enzyme. A major change in the enzyme takes place as protonation nears the pH optimum, this is recorded as a change in the enzyme's intrinsic affinity for malate (K_{m pH6.7} = 9.2 millimolar, K_{m pH7.7} = 28.3 millimolar). Similar changes in K_m have been observed for the NAD malic enzyme as it shifts from dimer to tetramer. It is most likely that the third ionizable group (probably a cysteine) revealed by the V_max/K_m profile is needed for optimal activity and is involved in the association-dissociation behavior of the enzyme.     

A nitrilotriacetic acid monooxygenase with conditional NADH-oxidase activity.

A tertiary amine monoxygenase from a Pseudomonas sp. was partially purified (35-fold) and characterized. In the presence of nitrilotriacetate (NTA), O₂, NADH, and Mn²⁺, the enzyme yielded two sets of products: iminodiacetate, glyoxylate, NAD⁺ and H₂O; or H₂O₂ and NAD⁺. Which set of products predominated was a function of enzyme concentration, ionic strength of solution, pH, and cation supplied. NTA functioned both as a modifiable substrate and as a stimulator of NADH oxidase activity. A requirement for preincubation with Mn²⁺ and NTA to eliminate enzyme hysteresis and the similar Km values for NTA and Mn²⁺ suggested that the substrate and metal were bound as a unit by the enzyme.     

Characterization of Mg2+-ATPase from sheep kidney medulla: purification.

Magnesium-dependent adenosine triphosphatase has been purified from sheep kidney medulla plasma membranes. The purification, which is based on treatment of a kidney plasma membrane fraction with 0.5% digitonin in 3 mm MgCl₂, effectively separates the Mg²⁺-ATPase from (Na⁺ + K⁺)-ATPase present in the same tissue and yields the Mg²⁺-ATPase in soluble form. The purified enzyme is activated by a variety of divalent cations and trivalent cations, including Mg²⁺, Mn²⁺, Ca²⁺, Co²⁺, Fe²⁺, Zn²⁺, Eu³⁺, Gd³⁺, and VO²⁺. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme shows two bands with R_f values corresponding to molecular weights of 150,000 and 77,000. The larger peptide is phosphorylated by [γ-³²P]ATP, suggesting that this peptide may contain the active site of the Mg²⁺-ATPase. The Mg²⁺-ATPase activity is unaffected by the specific (Na⁺ + K⁺)-ATPase inhibitor ouabain.     

Purification and characterization of pyruvate dehydrogenase complex from borccoli floral buds.

The pyruvate dehydrogenase complex (PDC) was purified from Brassica oleracea var. italica floral buds to a specific activity of approximately 6 μmol of NADH formed/min/ mg of protein. The PDC had cofactor requirements for NAD⁺, thiamine pyrophosphate, coenzyme A, and a divalent cation (Mg²⁺, Ca²⁺, or Mn²⁺). The enzyme catalyzed the oxidative decarboxylation of pyruvate at a rate threefold faster than 2-oxobutyrate but was inactive toward 2-oxoglutarate. The PDC was competively inhibited by acetyl-CoA against CoA and NADH against NAD⁺. The enzyme was shown to be more sensitive to regulation by NADH than acetyl-CoA.     

NADPH production from NADP+ using malic enzyme of Achromobacter parvulus IFO-13182

A sonicate of Achromobacter parvulus IFO-13182 produced NADPH from NADP⁺by an NADP⁺-linked malic enzyme [l-malate: NAD(P)⁺oxidoreductase, EC 1.1.1.39–40] reaction in the presence of l-malic acid and divalent metal ions. Malic enzyme of A. parvulus was stabilized by 5% l-malic acid, and activity was maintained at 60°C for 1 h. Contaminating phosphatase (orthophosphoricmonoester phosphohydrolase, EC 3.1.3.1–2) was completely inactivated by this treatment. Among the conditions tested, the optimum NADPH production was done using 36 μmol NADP⁺, 67 μmol l-malic acid, 63 μmol MgCl₂ and 1 unit of the malic enzyme in 3 ml of 55 mm phosphate buffer (pH 7.8). Conversion ratio of NADPH from NADP⁺ reached 100% after 4 h incubation at 30°C and the amount of NADPH accumulated was ～12 μmol ml^?1of the reaction mixture. No dephosphorylation of NADP⁺to NAD⁺or of NADPH to NADH was found by high performance liquid chromatography. The NADPH produced by such enzymatic reduction was purified by ethanol precipitation and dried in vacuo in powdered form with 97% purity, judged from the ratio of the absorbances at 340 and 260 nm. The purity of the NADPH produced was determined to be 95% from its coenzyme activity with NAD(P)⁺-linked glutathione reductase [NAD(P)H: oxidized-glutathione oxidoreductase, EC 1.6.4.2].     

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%.     

Partial Purification and Characterization of NADP-Isocitrate Dehydrogenase from Immature Pod Walls of Chickpea (Cicer arietinum L.)

NADP⁺-isocitrate dehydrogenase (threo-DS-isocitrate: NADP⁺ oxidoreductase [decarboxylating]; EC 1.1.1.42) (IDH) from pod walls of chickpea (Cicer arietinum L.) was purified 192-fold using ammonium sulfate fractionation, ion exchange chromatography on DEAE-Sephadex A-50, and gel filtration through Sephadex G-200. The purified enzyme, having a molecular weight of about 126,000, exhibited a broad pH optima from 8.0 to 8.6. It was quite stable at 4°C and had an absolute requirement for a divalent cation, either Mg²⁺ or Mn²⁺, for its activity. Typical hyperbolic kinetics was obtained with increasing concentrations of NADP⁺, dl-isocitrate, Mn²⁺, and Mg²⁺. Their K_m values were 15, 110, 15, and 192 micromolar, respectively. The enzyme activity was inhibited by sulfhydryl reagents. Various amino acids, amides, organic acids, nucleotides, each at a concentration of 5 millimolar, had no effect on the activity of the enzyme. The activity was not influenced by adenylate energy charge but decreased linearly with increasing ratio of NADPH to NADP⁺. Initial velocity studies indicated kinetic mechanism to be sequential. NADPH inhibited the forward reaction competitively with respect to NADP⁺ at fixed saturating concentration of isocitrate, whereas 2-oxoglutarate inhibited the enzyme noncompetitively at saturating concentrations of both NADP⁺ and isocitrate, indicating the reaction mechanism to be random sequential. Results suggest that the activity of NADP⁺-IDH in situ is likely to be controlled by intracellular NADPH to NADP⁺ ratio as well as by the concentration of various substrates and products.