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1.
Sterile cultures of Lemna minor grown in the presence of either nitrate, ammonium or amino acids failed to show significant changes in glutamate dehydrogenase (GDH) levels in response to nitrogen source. Crude and partially purified GDH preparations exhibit NADH and NADPH dependent activities. The ratio of these activities remain ca 12:1 during various treatments. Mixed substrate and product inhibition studies as well as electrophoretic behaviour suggest the existence of a single enzyme which is active in the presence of both coenzymes. GDH activity was found to be localized mainly in mitochondria. Kinetic studies revealed normal Michaelis kinetics with most substrates but showed deviations with NADPH and glutamate. A Hill-coefficient of 1.9 determined with NADPH indicates positive cooperative interactions, whereas a Hill-coefficient of 0.75 found with glutamate may be interpreted in terms of negative cooperative interactions. NADH dependent activity decreases rapidly during gel filtration whereas the NAD+ and NADPH activities remain unchanged. GDH preparations which have been pretreated with EDTA show almost complete loss of NADH and NAD+ activities. NADPH activity again remains unaffected. NAD+ activity is fully restored by adding Ca2+ or Mg2+, whereas the NADH activity can only be recovered by Ca2+ but not at all by Mg2+. Moderate inhibition of GDH reactions observed with various adenylates are fully reversed by adding Ca2+, indicating that the adenylate inhibition is due solely to the chelating properties of these compounds.  相似文献   

2.
Glutamate dehydrogenase, GDH (l-glutamate: NAD+ oxidoreductase (deaminating) EC 1.4.1.2) was purified from the plant fraction of lupin nodules and the purity of the preparation established by gel electrophoresis and electrofocusing. The purified enzyme existed as 4 charge isozymes with a MW of 270000. The subunit MW, as determined by dodecyl sulphate electrophoresis, was 45 000. On the basis of the results of the MW determinations a hexameric structure is proposed for lupin-nodule GDH. The pH optima for the enzyme were pH 8.2 for the amination reaction and pH 8.8 for the deamination reaction. GDH from lupin nodules showed a marked preference for NADH over NADPH in the amination reaction and used only NAD+ for the deamination reaction. Pyridoxal-5′-P and EDTA inhibited activity. The enzyme displayed Michaelis-Menten kinetics with respect to all substrates except NAD+. When NAD+ was the varied substrate, there was a deviation from Michaelis-Menten behaviour towards higher activity at high concentrations of NAD+.  相似文献   

3.
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.  相似文献   

4.
The H+-translocating inorganic pyrophosphatase (H+-PPase) associated with vesicles of the vacuolar membrane (tonoplast) isolated from beet (Beta vulgaris L.) is subject to direct inhibition by Ca2+ and a number of other divalent cations (Co2+, Mn2+, Zn2+). By contrast, the H+-translocating ATPase (H+-ATPase) located on the same membrane is insensitive to Ca2+. Here we examine the mechanism and feasibility of regulation of the vacuolar H+-PPase by cytosolic free Ca2+ under the conditions thought to prevail in vivo with respect to Mg2+, inorganic pyrophosphate (PPi), and pH. The minimal reaction scheme that satisfactorily describes the effects of elevated Ca2+ or CaPPi on the enzyme is one that invokes equilibrium binding of substrate (Mg2PPi) at one site, inhibitory binding of Mg2PPi to a lower-affinity second site, binding of activator (Mg2+) at a third site, and direct binding of Ca2+ or CaPPi to a fourth site. Changes in enzyme activity in response to selective manipulation of either Ca2+ or CaPPi are explicable only if Ca2+, rather than CaPPi, is the inhibitory ligand. This conclusion is supported by the finding that CaPPi fails to mimic substrate in protection of the enzyme from inhibition by N-ethylmaleimide. Furthermore, the reaction scheme quantitatively and independently predicts the observed noncompetitive effects of free Ca2+ on the substrate concentration dependence of H+-PPase activity. The results are discussed in relation to the previous proposal that CaPPi is the principal inhibitory ligand of the vacuolar H+-PPase (M. Maeshima [1991] Eur J Biochem 196: 11-17) and the possibility that in vivo modulation of cytosolic free Ca2+ might constitute a specific mechanism for selective regulation of this enzyme, and consequently for stabilization of PPi levels in the cytoplasm of plant cells.  相似文献   

5.
The pyruvate dehydrogenase complex was isolated from the mitochondria of broccoli florets and shown to be similar in its reaction mechanism to the complexes from other sources. Three families of parallel lines were obtained for the initial velocity patterns, indicating a multisite ping-pong mechanism. The apparent Km values obtained were 321 ± 18, 148 ± 13, and 7.2 ± 0.51 μm for pyruvate, NAD+, and CoA, respectively. Product inhibition studies using acetyl-CoA and NADH yielded results which were in agreement with those predicted by the multisite ping-pong mechanism. Acetyl-CoA and NADH were found to be competitive inhibitors versus CoA and NAD+, respectively. All other substrate-product combinations showed uncompetitive inhibition patterns, except for acetyl-CoA versus NAD+. Among various metabolites tested, only hydroxypyruvate (Ki = 0.11 mM) and glyoxylate (Ki = 3.27 mM) were found to be capable of inhibiting the broccoli enzyme to a significant degree. Initial velocity patterns using Mg2+? or Ca2+-thiamine pyrophosphate and pyruvate as the variable substrate were found to be consistent with an equilibrium ordered mechanism where Mg? or Ca-thiamine pyrophosphate bind first, with dissociation constants of 33.8 and 3 μm, respectively. The Mg- or Ca-thiamine pyrophosphate complexes also dissociated rapidly from the enzyme complex.  相似文献   

6.
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 malate2? at a saturating concentration of Mg2+ or by varying Mg2+ and NAD+ at a saturating level of malate2? 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 malate2? 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 Vmax or Km,malate2? but the Km for metal and NAD+ are greatly decreased. At pH 7.0 and above, Mn2+ isotherms and malate2? 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.  相似文献   

7.
The amination of α-ketoglutarate (α-KG) by NADH-glutamate dehydrogenase (GDH) obtained from Sephadex G-75 treated crude extracts from shoots of 5-day-old seedlings was stimulated by the addition of Ca2+. The NADH-GDH purified 161-fold with ammonium sulfate, DEAE-Toyopearl, and Sephadex G-200 was also activated by Ca2+ in the presence of 160 micromolar NADH. However, with 10 micromolar NADH, Ca2+ had no effect on the NADH-GDH activity. The deamination reaction (NAD-GDH) was not influenced by the addition of Ca2+.

About 25% of the NADH-GDH activity was solubilized from purified mitochondria after a simple osmotic shock treatment, whereas the remaining 75% of the activity was associated with the mitochondrial membrane fraction. When the lysed mitochondria, mitochondrial matrix, or mitochondrial membrane fraction was used as the source of NADH-GDH, Ca2+ had little effect on its activity. The mitochondrial fraction contained about 155 nanomoles Ca per milligram of mitochondrial protein, suggesting that the NADH-GDH in the mitochondria is already in an activated form with regard Ca2+. In a simulated in vitro system using concentrations of 6.4 millimolar NAD, 0.21 millimolar NADH, 5 millimolar α-KG, and 5 millimolar glutamate thought to occur in the mitochondria, together with 1 millimolar Ca2+, 10 and 50 millimolar NH4+, and purified enzyme, the equilibrium of GDH was in the direction of glutamate formation.

  相似文献   

8.
Mitochondria from the parasitic helminth, Hymenolepis diminuta, catalyzed both NADPH:NAD+ and NADH:NADP+ transhydrogenase reactions which were demonstrable employing the appropriate acetylpyridine nucleotide derivative as the hydride ion acceptor. Thionicotinamide NAD+ would not serve as the oxidant in the former reaction. Under the assay conditions employed, neither reaction was energy linked, and the NADPH:NAD+ system was approximately five times more active than the NADH:NADP+ system. The NADH:NADP+ reaction was inhibited by phosphate and imidazole buffers, EDTA, and adenyl nucleotides, while the NADPH:NAD+ reaction was inhibited only slightly by imidazole and unaffected by EDTA and adenyl nucleotides. Enzyme coupling techniques revealed that both transhydrogenase systems functioned when the appropriate physiological pyridine nucleotide was the hydride ion acceptor. An NADH:NAD+ transhydrogenase system, which was unaffected by EDTA, or adenyl nucleotides, also was demonstrable in the mitochondria of H. diminuta. Saturation kinetics indicated that the NADH:NAD+ reaction was the product of an independent enzyme system. Mitochondria derived from another parasitic helminth, Ascaris lumbricoides, catalyzed only a single transhydrogenase reaction, i.e., the NADH:NAD+ activity. Transhydrogenase systems from both parasites were essentially membrane bound and localized on the inner mitochondrial membrane. Physiologically, the NADPH:NAD+ transhydrogenase of H. diminuta may serve to couple the intramitochondrial metabolism of malate (via an NADP linked “malic” enzyme) to the anaerobic NADH-dependent ATP-generating fumarate reductase system. In A. lumbricoides, where the intramitochondrial metabolism of malate depends on an NAD-linked “malic” enzyme which is localized primarily in the intermembrane space, the NADH:NAD+ transhydrogenase activity may serve physiologically in the translocation of hydride ions across the inner membrane to the anaerobic energy-generating fumarate reductase system.  相似文献   

9.
Effect of polyamines on the methylation of adenine in 16S rRNA was examined using the purified methylating enzyme. When 23S core particles were used as substrate, the activity was stimulated by Mg2+, Ca2+ and monovalent cations. Even in the presence of optimal concentrations of Mg2+ and NH4+, the addition of 1 mM spermidine stimulated the methylation approximately 1.7-fold. When 30S ribosomal subunits were used as substrate, the rate of methylation was 20% of that of the methylation of 23S core particles. The activity was not influenced significantly by Mg2+, Ca2+ or monovalent cations. The addition of spermidine inhibited the methylation.  相似文献   

10.
目的:研究兔肌3-磷酸甘油脱氢酶的分离纯化方法及其酶学性质,为测定血清甘油三酯所用酶联试剂的开发提供试验基础和理论依据。方法:通过硫酸铵分级沉淀、DEAE-Sepharose、Blue-Sepharose和羟磷灰石纯化兔肌3-磷酸甘油脱氢酶,利用凝胶过滤和梯度PAGE(5%~15%)法测定酶分子量,采用常规酶学动力学分析方法,考察pH、温度、底物浓度以及部分金属离子与有机化合物对酶促反应的影响。结果 纯化后的兔肌3-磷酸甘油脱氢酶经PAGE(12%)分析为单一条带;酶分子量为115~122 kDa;酶最适温度45℃,最适pH 9;酸碱稳定范围pH6~9,低于45℃时热稳定性好;最适条件下,以3-磷酸甘油和NAD+为底物,测得酶的Km分别为7.4×10-3mol/L和1.47×10-4mol/L;Ba2+、Mn2+、Fe2+、Al3+、Cu2+、Ni2+、Ag+、Hg2+、NaN3、EDTA对酶有不同程度的抑制作用,Mg2+、Ca2+、Co2+、Zn2+有一定程度的激活作用,其中Co2+和Zn2+对酶的激活作用能达到200%以上,有机化合物NaF对酶的活性没有影响。  相似文献   

11.
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.  相似文献   

12.
The influence of selected factors on the activity of highly purified GDH in triticale roots was investigated in vitro. In the presence of 2-ME, NADH-GDH activity increased by 400 %, while NADPH-GDH activity rose by 500 %. No effect of reducing factors on NAD(P)+-GDH reaction was detected. The sulphydryl groups inhibitors, such as p-chloromercuribenzoate (p-CMB) and iodoacetamide, proved the strongest inhibitors of the aminative reaction. Metal-binding compounds: ethylenediaminetetraacetic acid disodium salt (EDTA) and Zinkov also considerably inhibited NAD(P)H-GDH activity. Diisopropylfluorophosphate (DFP) and pepstatin A, the inhibitors specific for -OH serine and COO aspartic acid groups respectively, caused a non-significant NAD(P)H-GDH activity decrease. Cd2+, Co2+, Hg2+, Mg2+, Pb2+ and Zn2+ ions strongly inhibited the amination reaction, whereas their inhibiting effect upon NAD+-GDH activity was negligible. Among the applied ions, only Ca2+ activated NADH-GDH.  相似文献   

13.
The reaction of glutamate dehydrogenase (l-glutamate: NAD+ oxidoreductase (deaminating) EC 1.4.1.2) from lupin nodules has been investigated in the direction of deamination by means of steady state velocity studies in the absence of products and inhibition studies with products and substrate analogs. The results are qualitatively and quantitatively consistent with a fully ordered reaction mechanism in which NAD+ binds to the enzyme first followed by l-glutamate. The order of product release is proposed to be NH4+ followed by 2-oxoglutarate and then NADH. In addition, product inhibition data indicate the formation of an enzyme-NAD-oxoglutarate dead-end complex.  相似文献   

14.
Bush LP 《Plant physiology》1969,44(3):347-350
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.  相似文献   

15.
Activities and some properties of microsomal ATPases have been studied in developing human placenta. The enzyme activities (Na+ + K+ + Mg2+, Mg2+, and Ca2+ dependent) in the placenta increase steadily with gestational age until the 18th to 21st week, and decrease in the second half of pregnancy. Mg2+-dependent and Na+ + K+ + Mg2+-dependent ATPases possess nearly the same Km (apparent) for ATP, while the Ca2+-dependent enzyme shows a different one. Mg2+-dependent ATPase shows higher substrate affinity than Ca2+-dependent ATPase, although the Vmax of the Mg2+-dependent enzyme is lower than that of the latter. However, for each enzyme, the Km remains almost constant and Vmax varies during ontogenic development. Vmax of the enzymes decline at term. The enzymes are heat-labile, unaffected by amino acids, namely, l-phenylalanine, l-leucine, and l-tryptophan, and deoxycholate inhibits the enzyme activities by about 50%.  相似文献   

16.
Tb3+, a fluorescent trivalent cation with physicochemical properties similar to Ca2+, binds to peripheral nerve membrane vesicles prepared from the walking leg nerve bundle of the lobster (Homarus americanus). Saturable binding is measured for at least two classes of binding site. Bound Tb3+ can be displaced by other cations in the order: Ca2+ > Mg2+ = Zn2+ > NH4+. The binding of Tb3+ to the lower affinity site (KD(app) = 6.0 μM) is inhibitable by Na+, Mg2+ and Ca2+, whereas the higher affinity site (KD(app) = 2.2 μM) is only sensitive to Ca2+. Using this spectral probe the role of Ca2+ in peripheral nerve membrane function can be investigated.  相似文献   

17.
The cardiac Na+/Ca2+ exchanger (NCX) is the major Ca2+ efflux pathway on the sarcolemma, counterbalancing Ca2+ influx via L-type Ca2+ current during excitation-contraction coupling. Altered NCX activity modulates the sarcoplastic reticulum Ca2+ load and can contribute to abnormal Ca2+ handling and arrhythmias. NADH/NAD+ is the main redox couple controlling mitochondrial energy production, glycolysis, and other redox reactions. Here, we tested whether cytosolic NADH/NAD+ redox potential regulates NCX activity in adult cardiomyocytes. NCX current (INCX), measured with whole cell patch clamp, was inhibited in response to cytosolic NADH loaded directly via pipette or increased by extracellular lactate perfusion, whereas an increase of mitochondrial NADH had no effect. Reactive oxygen species (ROS) accumulation was enhanced by increasing cytosolic NADH, and NADH-induced INCX inhibition was abolished by the H2O2 scavenger catalase. NADH-induced ROS accumulation was independent of mitochondrial respiration (rotenone-insensitive) but was inhibited by the flavoenzyme blocker diphenylene iodonium. NADPH oxidase was ruled out as the effector because INCX was insensitive to cytosolic NADPH, and NADH-induced ROS and INCX inhibition were not abrogated by the specific NADPH oxidase inhibitor gp91ds-tat. This study reveals a novel mechanism of NCX regulation by cytosolic NADH/NAD+ redox potential through a ROS-generating NADH-driven flavoprotein oxidase. The mechanism is likely to play a key role in Ca2+ homeostasis and the response to alterations in the cytosolic pyridine nucleotide redox state during ischemia-reperfusion or other cardiovascular diseases.  相似文献   

18.
Summary The control by nucleotides of the Ca2+-activated channel which regulates the nonspecific permeability of the mitochondrial inner membrane has been investigated quantitatively. The cooperative binding of two molecules of ADP to the internal (matrix) side of the channel causes a mixed-type inhibition of channel activity. ATP, AMP, cAMP and GDP are all ineffective. NADH shows a pattern of inhibition similar to that of ADP, though the apparentK I is higher by a factor of 200. NADPH relieves the inhibition by NADH. NAD+ also inhibits, but its affinity is a factor of 10 less than that of NADH. When ADP and NADH are added together, they act synergistically to inhibit the Ca2+-activated channel. It is concluded that the concept of the modification of enzyme activity by the allosteric binding of nucleotides, which is well established for soluble enzyme systems, also has application to the regulation of channels that control membrane permeability.  相似文献   

19.
An acidophilic and Ca2+-independent amylase was purified from a newly isolated Bacillus sp. DR90 by ion-exchange chromatography, and exhibited a molecular weight of 68.9 kDa by SDS-PAGE. The optimum pH and temperature of the enzyme were found to be 4.0 and 45 °C, respectively. The enzyme activity was increased by Ba2+, Fe2+ and Mg2+, and decreased by Hg2+ and Zn2+, while it was not affected by Na+, K+, phenylmethylsulfonyl fluoride and β-mercaptoethanol. Ca2+ and EDTA did not have significant effect on the enzyme activity and thermal stability. The values of K m and V max for starch as substrate were 4.5 ± 0.13 mg/ml and 307 ± 12 μM/min/mg, respectively. N,N-dialkylimidazolium-based ionic liquids such as 1-hexyl-3-methylimidazolium bromide [HMIM][Br] have inhibitory effect on the enzyme activity. Thin layer chromatography analyses displayed that maltose and glucose are the main products of the enzyme reaction on starch. Regarding the features of the enzyme, it may be utilized as a novel candidate for industrial applications.  相似文献   

20.
Several aspects of Mg2+ homeostasis were investigated in cultured chicken heart cells using the fluorescent Mg2+ indicator, FURAPTRA. The concentration of cytosolic Mg2+ ([Mg2+]i) is 0.48 ± 0.03 mM (n = 31). To test whether a putative Na/Mg exchange mechanism controls [Mg2+]i below electrochemical equilibrium, we manipulated the Na+ gradient and assessed the effects on [Mg2+]i. When extracellular Na+ was removed, [Mg2+]i increased; this increase was not altered in Mg-free solutions, but was attenuated in Ca-free solutions. A similar increase in [Mg2+]i, which was dependent upon extracellular Ca2+, was observed when intracellular Na+ was raised by inhibiting the Na/K pump with ouabain. These results do not provide evidence for Na/Mg exchange in heart cells, but they suggest that Ca2+ can modulate [Mg2+]i. In addition, removing extracellular Na+ caused a decrease in intracellular pH (pHi), as measured by pH-sensitive microelectrodes, and this acidification was attenuated when Cat+ was also removed from the solution. These results suggest that Ca2+ and H+ interact intracellularly. Since changes in the Na+ gradient can also alter pHi, we questioned whether pH can modulate [Mg2+]i. pHi was manipulated by the NH4Cl prepulse method. NH4 +-evoked changes in pHi, as measured by the fluorescent indicator BCECF, were accompanied by opposite changes in [Mg2+]i; [Mg2+]i changed by –0.16 mM/unit pH. These NH4 +-evoked changes in [Mg2+]i were not caused by movements of Mg2+ or Ca2+ across the sarcolemma or by changes in cytosolic Ca2+. Additionally, pHi was manipulated by changing extracellular pH (pHo). When pHo was decreased from 7.4 to 6.3, pHi decreased by 0.64 units and [Mg2+]i increased by 0.12 mM; in contrast, when pHo was raised from 7.4 to 8.3, pHi increased by 0.6 units and [Mg2+]i did not change significantly. The results of our investigations suggest that Ca 2+ and H+ can modulate [Mg2+]i, probably by affecting cytosolic Mg2+ binding and/or subcellular Mg2+ transport and that such redistribution of intracellular Mg2+ may play an important role in Mg2+ homeostasis in cardiac cells.  相似文献   

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