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1.
A plasma membrane-rich microsome fraction isolated from barley (Hordeum vulgare L. cv. Conquest) roots contained considerable divalent cation-dependent ATPase activity when assayed at 16°C. The maximal divalent cation-stimulation of the apparent basal ATPase activity varied as Ca2+ > Mg2+ > Mn2+= Zn2+ > Co2+ > Ni2+, with all other divalent cations tested being inhibitory. Double reciprocal plots of the Ca2+- and Mg2+-dependent ATPase velocities as a function of substance concentration were nonlinear, suggesting the presence of multiple catalytic sites. Both MgATP2- and CaATP2- served as the true substrates and apparently bind to the same catalytic sites. Free ATP and Ca2+ could inhibitit the Ca2+- and Mg2+-dependent ATPase. Increasing free Mg2+ levels enhanced the affinity of the Mg2+-dependent ATPase for MgATP2-, while slightly inhibiting the Vmax values. Other divalent cation-nucleoside triphosphate complexes produced maximal enzyme velocities equal to or greater than those generated by CaATP2- and MgATP2-. However, the ATPase had significantly higher affinities for CaATP2- and MgATP2-, than for the alternative substrates. The high and low affinity components of the Ca2+- and Mg2+-dependent ATPase exhibited optimal Vmax values at pH 5 and 6, respectively. Analysis of the pH-dependence of the enzyme Km values indicated enzyme-substrate binding with charge neutralization at neutral and alkaline pH's. Nonlinear double reciprocal plots were obtained at all assay temperatures. However, the complexity of the enzyme kinetics became less apparent at the higher assay temperatures. The kinetics of the barley root divalent cation-dependent ATPase activities are discussed in terms of the kinetics of ATPases from other plants and the methods used to obtain them, and compared to the kinetics of ion transport ATPases from animal membranes.  相似文献   

2.
The influences of total magnesium ion concentration at different total ATP concentrations, and of total ATP concentration, for different total magnesium ion concentrations, on the enzymatic rate of the isolated chloroplast F1 ATPase, have been followed by a chromatographic method consisting in the separation and determination of ADP. From the various series of curves, it is concluded that the experimental results (position of the maxima,K m values) are better fitted by a mechanism involving the activation of the enzyme by magnesium ion and hydrolysis of free ATP, rather than by the classical mechanism, for which the enzyme hydrolyzes the MgATP complex and is inhibited by Mg2+. Although the equations giving the reaction rate are similar in the two cases, the calculated values ofK m are widely different. The value obtained from the classical mechanism does not agree withK D , the dissociation constant of the enzyme-substrate complex, measured by the Hummel and Dreyer method. Moreover, when the total ATP concentration tends toward the total magnesium ion concentration, the nucleotide binding to the enzyme tends toward zero, although it should be maximum if MgATP were the true substrate. Finally, the inhibitory effect of Na+ is more easily explained as a competition between this ion and the activating Mg2+, than by the classical mechanism.  相似文献   

3.
Some aspects of theEscherichia coli Lon protease ATPase function were studied around the optimum pH value. It was revealed that in the absence of the protein substrate the maximum ATPase activity of the enzyme is observed at an equimolar ratio of ATP and Mg2+ ions in the area of their millimolar concentrations. Free components of the substrate complex (ATP-Mg)2− inhibit the enzyme ATPase activity. It is hypothesized that the effector activity of free Mg2+ ions is caused by the formation of the “ADP-Mg-form” of ATPase centers. It was shown that the activation of ATP hydrolysis in the presence of the protein substrate is accompanied by an increase in the affinity of the (ATP-Mg)2− complex to the enzyme, by an elimination of the inhibiting action of free Mg2+ ions without altering the efficiency of catalysis of ATP hydrolysis (based on thek cat value), and by a change in the type of inhibition of ATP hydrolysis by the (ADP-Mg) complex (without changing theK i value). Interaction of the Lon protease protein substrate with the enzyme area located outside the peptide hydrolase center was demonstrated by a direct experiment.  相似文献   

4.
Cytosolic ATP-phosphofructokinase (PFK) from spinach leaves (Spinacia oleracea L.) was inhibited by submillimolar concentrations of free Mg2+. The free Mg2+ concentration required for 50% inhibition of PFK activity was 0.22 millimolar. Inhibition by free Mg2+ was independent of the MgATP2− concentration. Inorganic phosphate (Pi) reduces the inhibition of PFK activity by Mg2+. Free ATP (ATP4−) also inhibits PFK activity. For free ATP the inhibition of PFK activity was dependent on the MgATP2− concentration. Fifty percent inhibition of PFK activity requires 1.2 and 3.7 millimolar free ATP at 0.1 and 0.5 millimolar MgATP2−, respectively. It was proposed that free ATP competes for the MgATP2− binding site, whereas free Mg2+ does not. Pi diminished the inhibitory effect of free ATP on PFK activity. Free ATP and Pi had substantial effects on the MgATP2− requirement of cytosolic PFK. For half-maximum saturation of PFK activity 3 and 76 micromolar MgATP2− was required at 0.007 and 0.8 millimolar free ATP in the absence of Pi. At 5 and 25 millimolar Pi, half-maximum saturation was achieved at 9 and 14 micromolar MgATP2−. PFK activity was inhibited by Ca2+. The inhibition by Ca2+ depends upon the total Mg2+ concentration. Fifty percent inhibition of PFK activity required 22 and 32 micromolar Ca2+ at 0.1 and 0.2 millimolar Mg2+, respectively. At physiological concentrations of about 0.5 millimolar free Mg2+, Ca2+ would have little effect on cytosolic PFK activity from spinach leaves. PFK is not absolutely specific for the nucleoside 5′-triphosphate substrate. Besides MgATP2−, MgUTP2−, MgCTP2−, and MgGTP2− could be used as a substrate. All four free nucleotides inhibit PFK activity. The physiological consequences of the regulatory properties of cytosolic PFK from spinach leaves will be discussed. A model will be introduced, in an attempt to describe the complex interaction of PFK with substrates and the effectors Mg2+ and Pi.  相似文献   

5.
The proton translocating membrane ATPase of oral streptococci has been implicated in cytoplasmatic pH regulation, acidurance and cariogenicity. Studies have confirmed that Streptococcus mutans is the most frequently detected species in dental caries. A P-type ATPase that can act together with F1Fo-ATPase in S. mutans membrane has been recently described. The main objective of this work is to characterize the kinetic of ATP hydrolysis of this P-type ATPase. The optimum pH for ATP hydrolysis is around 6.0. The dependence of P-type ATPase activity on ATP concentration reveals high (K0.5=0.27 mM) and low (K0.5=3.31 mM) affinity sites for ATP, exhibiting positive cooperativity and a specific activity of about 74 U/mg. Equimolar concentrations of ATP and magnesium ions display a behavior similar to that described for ATP concentration in Mg2+ saturating condition (high affinity site, K0.5=0.10 mM, and low affinity site, K0.5=2.12 mM), exhibiting positive cooperativity and a specific activity of about 68 U/mg. Sodium, potassium, ammonium, calcium and magnesium ions stimulate the enzyme, showing a single saturation curve, all exhibiting positive cooperativities, whereas inhibition of ATPase activity is observed for zinc ions and EDTA. The kinetic characteristics reveal that this ATPase belongs to type IIIA, like the ones found in yeast and plants.  相似文献   

6.
Ion stimulation and some other properties of an ATPase activity associated with vacuoles isolated from storage roots of red beet (Beta vulgaris L.) have been determined. The ATPase had a specific requirement for Mg2+ and in the presence of Mg2+ it was stimulated by salts of monovalent cations. The degree of stimulation by monovalent salts was influenced mainly by the anion and the order of effectiveness of the anions tested was Cl->HCO 3 - >Br->malate>acetate>SO 4 2- . For any given series of anions the magnitude of the stimulation obtained was influenced by the accompanying cation (NH 4 + Na+>K+). This cation effect was abolished by 0.01% (v/v) Triton X-100 and it is suggested that it is the result of different permeabilities of membrane vesicles to the cations. There was no evidence of synergistic stimulation of the ATPase by mixtures of Na+ and K+. KCl- and NaCl-stimulation was maximal with salt concentrations in the range 60–150 mM. The true substrate of the enzyme was shown to be MgATP. It was shown that KCl stimulation was the result of an increase in Vmax rather than a change in the affinity of the enzyme for MgATP. The ATPase was inhibited by N,N-dicyclohexylcarbodiimide, diethylstilbestrol, mersalyl and KNO3 but other inhibitors tested (azide, oligomycin, orthovanadate, K3[Cr(oxalate)6] and ethyl-3-[3-dimethylaminopropyl]carbodiimide) were without effect or caused only partial inhibition at the highest concentration tested. The ATPase activity was equally distributed between pellet and supernatant fractions obtained after the subfractionation of vacuoles but the properties of the ATPase in each fraction were the same. It is suggested that beet vacuoles possess only one ATPase. The properties of the ATPase are compared with those of ATPases associated with other plant membranes and organelles and its possible role in transport at the tonoplast is discussed.Abbreviations ATPF free ATP - ATPT total ATP - BSA bovine serum albumen - DCCD N,N-dicyclohexylcarbodiimide - DES diethylstilbestrol - DNP 2,4-dinitrophenol - EDAC ethyl-3-(3-dimethylaminopropyl)carbodiimide - Km apparent Michaelis constant - MgATP complex of Mg2+ and ATP - Mg F 2+ free Mg2+ - Mg T 2 total Mg2+ - MES 2-(N-Morpholino)ethanesulphonic acid - Na2EDTA disodium ethylenediaminetetraacetic acid - NEM N-ethylmaleimide - Pi inorganic phosphate - TCA trichloroacetic acid - Tris tris(hydroxymethyl)methylamine - Vmax maximum velocity  相似文献   

7.
Isolated hepatocytes converted exogenous [α-32P]ATP to cyclic [32P]AMP at high rates. This system was used for kinetic studies of the effects of glucagon, fluoride, free magnesium and free ATP4? on adenylate cyclase. In the absence or presence of glucagon, free Mg2+ activated adenylate cyclase by decreasing the Km for MgATP2? without changing V. Free ATP4? was not a potent inhibitor of adenylate cyclase and the only effect of glucagon was to increase V.Fluoride also increased the V of adenylate cyclase, but, in contrast to the results obtained with glucagon, the effect increased as the concentration of free Mg2+ increased. One explanation of the effect of fluoride, consistent with the idea that free Mg2+ activates adenylate cyclase and free ATP is not an inhibitor, is that fluoride increases the affinity of the enzyme for Mg2+. Weak inhibition of adenylate cyclase by ATP4? in the presence of fluoride cannot be excluded.  相似文献   

8.
Two distinct membrane fractions containing H+-ATPase activity were prepared from red beet. One fraction contained a H+-ATPase activity that was inhibited by NO3 while the other contained a H+-ATPase inhibited by vanadate. We have previously proposed that these H+-ATPases are associated with tonoplast (NO3-sensitive) and plasma membrane (vanadate-sensitive), respectively. Both ATPase were examined to determine to what extent their activity was influenced by variations in the concentration of ATPase substrates and products. The substrate for both ATPase was MgATP2−, and Mg2+ concentrations in excess of ATP had only a slight inhibitory effect on either ATPase. Both ATPases were inhibited by free ATP (i.e. ATP concentrations in excess of Mg2+) and ADP but not by AMP. The plasma membrane ATPase was more sensitive than the tonoplast ATPase to free ATP and the tonoplast ATPase was more sensitive than the plasma membrane ATPase to ADP.

Inhibition of both ATPases by free ATP was complex. Inhibition of the plasma membrane ATPase by ADP was competitive whereas the tonoplast ATPase demonstrated a sigmoidal dependence on MgATP2− in the presence of ADP. Inorganic phosphate moderately inhibited both ATPases in a noncompetitive manner.

Calcium inhibited the plasma membrane but not the tonoplast ATPase, apparently by a direct interaction with the ATPase rather than by disrupting the MgATP2− complex.

The sensitivity of both ATPases to ADP suggests that under conditions of restricted energy supply H+-ATPase activity may be reduced by increases in ADP levels rather than by decreases in ATP levels per se. The sensitivity of both ATPases to ADP and free ATP suggests that modulation of cytoplasmic Mg2+ could modulate ATPase activity at both the tonoplast and plasma membrane.

  相似文献   

9.
10.
In contrast to everted mitochondrial inner membrane vesicles and eubacterial plasma membrane vesicles, the ATPase activity of chloroplast ATP synthase in thylakoid membranes is extremely low. Several treatments of thylakoids that unmask ATPase activity are known. Illumination of thylakoids that contain reduced ATP synthase (reduced thylakoids) promotes the hydrolysis of ATP in the dark. Incubation of thylakoids with trypsin can also elicit higher rates of ATPase activity. In this paper the properties of the ATPase activity of the ATP synthase in thylakoids treated with trypsin are compared with those of the ATPase activity in reduced thylakoids. The trypsin-treated membranes have significant ATPase activity in the presence of Ca2+, whereas the Ca2+-ATPase activity of reduced thylakoids is very low. The Mg2+-ATPase activity of the trypsinized thylakoids was only partially inhibited by the uncouplers, at concentrations that fully inhibit the ATPase activity of reduced membranes. Incubation of reduced thylakoids with ADP in Tris buffer prior to assay abolishes Mg2+-ATPase activity. The Mg2+-ATPase activity of trypsin-treated thylakoids was unaffected by incubation with ADP. Trypsin-treated membranes can make ATP at rates that are 75–80% of those of untreated thylakoids. The Mg2+-ATPase activity of trypsin-treated thylakoids is coupled to inward proton translocation and 10 mM sulfite stimulates both proton uptake and ATP hydrolysis. It is concluded that cleavage of the γ subunit of the ATP synthase by trypsin prevents inhibition of ATPase activity by the ε subunit, but only partially overcomes inhibition by Mg2+ and ADP during assay.  相似文献   

11.
The properties of membrane-associated ATPase of cucumber (Cucumis sativus cv. Seiriki No. 2) roots cultured in a complete medium (complete enzyme) and in a medium lacking Ca2+ (Ca2+-deficient enzyme) were investigated. The basal activity of membrane-associated ATPase increased during Ca2+ starvation, while Mg2+-activation of the enzyme decreased and even resulted in inhibition by high Mg2+ concentration at the late stage of the Ca2+ starvation. The complete enzyme had low basal activity and showed a Mg2+-activated hyperbolic reaction curve in relation to ATP concentration. Ca2+-deficient enzyme with high basal activity showed a biphasic reaction curve and Mg2+-activation was seen only at high ATP concentrations. Activation of membrane-associated ATPase by various cations was decreased or lost during Ca2+ starvation. The basal ATPase activity of Ca2+-deficient enzyme increased for various substrates including pyrophosphate, p-nitrophenyl phosphate, glucose-6 phosphate, β-glycerophosphate, AMP, ADP and ATP. Mg2+-activation was found only for ADP and ATP in both the complete and Ca2+-deficient enzymes, but the activation for ATP was greatly reduced by Ca2+ starvation. The heat inactivation curves for basal and Mg2+-activated ATPase did not differ much between the complete and Ca2+-deficient enzyme. The delipidation of membrane-associated enzyme by acetone affected the protein content and the basal activity slightly, but inhibited the Mg2+-activated ATPase activity clearly with somewhat different behaviour between the complete and Ca2+-deficient enzyme.  相似文献   

12.
The plasma membrane Ca2+ ATPase catalyzed the hydrolysis of ATP in the presence of millimolar concentrations of EGTA and no added Ca2+ at a rate near 1.5% of that attained at saturating concentrations of Ca2+. Like the Ca-dependent ATPase, the Ca-independent activity was lower when the enzyme was autoinhibited, and increased when the enzyme was activated by acidic lipids or partial proteolysis. The ATP concentration dependence of the Ca2+-independent ATPase was consistent with ATP binding to the low affinity modulatory site. In this condition a small amount of hydroxylamine-sensitive phosphoenzyme was formed and rapidly decayed when chased with cold ATP. We propose that the Ca2+-independent ATP hydrolysis reflects the well known phosphatase activity which is maximal in the absence of Ca2+ and is catalyzed by E2-like forms of the enzyme. In agreement with this idea pNPP, a classic phosphatase substrate was a very effective inhibitor of the ATP hydrolysis.  相似文献   

13.
The effect of pH and of Mg2+ concentration on the light activated form of stromal fructose-1,6-bisphosphatase (FBPase) was studied using the enzyme rapidly extracted from illuminated spinach chloroplasts. The (fructose-1,6-bisphosphate4-)(Mg2+) complex has been identified as the substrate of the enzyme. Therefore, changes of pH and Mg2+ concentrations have an immediate effect on the activity of FBPase by shifting the pH and Mg2+ dependent equilibrium concentration of the substrate. In addition, changes of pH and Mg2+ concentration in the assay medium have a delayed effect on FBPase activity. A correlation of the activities observed using different pH and Mg2+ concentrations indicates, that the effect is not a consequence of the pH and Mg2+ concentration as such, but is caused by a shift in the equilibrium concentration of a hypothetical inhibitor fructose-1,6-bisphosphate3- (uncomplexed), resulting in a change of the activation state of the enzyme. The interplay between a rapid effect on the concentration of the substrate and a delayed effect on the activation state enables a rigid control of stromal FBPase by stromal Mg2+ concentrations and pH. Fructose-1,6-bisphosphatase is allosterically inhibited by fructose-6-phosphate in a sigmoidal fashion, allowing a fine control of the enzyme by its product.Abbreviations Fru1,6 bis P fructose-1,6-bisphosphate - Fru6P fructose-6-phosphate - FBPase fructose-1,6-bisphosphatase Some of these results have been included in a preliminary report (Heldt et al. 1984)  相似文献   

14.
Luit Slooten  Adriaan Nuyten 《BBA》1981,638(2):313-326
(1) Light-activated ‘dark’ ATPase in Rhodospirillum rubrum chromatophores is inhibited by preincubation with ADP or ATP (in the absence of Mg2+). I50 values were 0.5 and 6 μM, respectively, after 20 s of preincubation. (2) In the absence of MgATP, the rate constant for dissociation of ADP or ATP from the inhibitory site was less than 0.2 min?1 in deenergized membranes. Illumination in the absence of MgATP caused an increase of over 60-fold in both rate constants. (3) In some experiments hydrolysis was performed in the presence of 10 μM Mg2+ and 0.2 mM MgATP. Under these conditions, the ADP or ATP inhibition was reversed within about 20 or about 80 s, respectively, after the onset of hydrolysis. This suggests that recovery from ADP or ATP inhibition (i.e., release of tightly bound ADP or ATP) in the dark is induced by MgATP binding to a second nucleotide-binding site on the enzyme. (4) Results obtained with variable concentrations of uncoupler suggest that in the absence of bound Mg2+ (see below), MgATP-induced release of tightly bound ADP or ATP does not require a transmembrane Δ\?gmH+. This, together with the inhibitor/substrate ratios prevalent during hydrolysis, suggests that these reactivation reactions involve MgATP binding to a high-affinity binding site (Kd < 2 μM). (5) At high concentrations of uncoupler, a time-dependent inhibition of hydrolysis occurred in the control chromatophores as well as in the nucleotide-pretreated chromatophores. This deactivation was dependent on Mg2+. In addition, MgATP-dependent reversal of ADP inhibition in the dark was inhibited by Mg2+ at concentrations above 20–30 μM. By contrast, MgATP-dependent reversal of ADP inhibition occurs within 3–4 s, despite the presence of high concentrations of Mg2+ if the chromatophores are illuminated during contact with the nucleotides. Uncoupler abolishes the effect of illumination. A reaction scheme incorporating these findings is proposed. (6) The implications of these findings for the mechanism of lightactivation of ATP hydrolysis (Slooten, L. and Nuyten, A., (1981) Biochim. Biophys. Acta 638, 305–312) are discussed.  相似文献   

15.
 The membrane-bound F1 sector of the H+–ATPase complex (F-type ATPase) in dark-adapted photosynthetic chromatophores is endowed with MgATP- and CaATP-dependent ATPase activities, both sensitive to inhibitors such as oligomycin and venturicidin. Because of contatamination of free Mg2 + and Ca2+ ions in chromatophore preparations, kinetic characterization of the two hydrolitic reactions can be performed only in the presence of both substrates, using a model for two alternative substrates. The two activities are characterized by similar maximal rates and affinity constants [VMgATP and VCaATP: 13±1 and 10±1 nmol s–1 ATP hydrolyzed (μmol BChl)–1; KMgATP and KCaATP: 0.22±0.06 and 0.20±0.05 mm]. However, only the MgATP-dependent ATPase is coupled to Δ*H + generation. In this process CaATP acts as an alternative substrate and a competitive inhibitor of the proton pump, with a KI coincident with KCaATP for the hydrolytic activity. This finding highlights the central role that the coordination chemistry of the ion-nucleotide complex plays in determining the proton gating mechanism at the catalytic site(s) of the enzyme complex. These results are discussed on the basis of the coordination properties of the ions and of the available information on the protein structure. Received: 5 December 1995 / Accepted: 7 March 1996  相似文献   

16.
《FEBS letters》1985,188(2):379-382
Magnesium-induced enzymatic and structural changes of membrane-bound proteins in rat heart sarcolemma have been investigated. In the absence of ATP, increasing concentrations of magnesium within the range 0.1–10.0 mM gradually lowered the α-helix content of sarcolemmal proteins. The same magnesium concentrations stepwise activated the Mg2+-dependent ATPase in the presence of ATP. Mathematical and graphical analysis of the data yielded a quantitative relationship between magnesium-induced stimulation of the Mg2+-dependent ATPase activity and diminution of the α-helix content of membrane proteins in cardiac sarcolemma.  相似文献   

17.
We have studied the activities of Ca2+-stimulated ATPase in rat heart sarcolemma upon modulating the redox state of membrane thiol groups with dithiothreitol (DTT). The suitability of alamethicin to unmask the latent activity of this enzyme was also investigated. The Ca2+-stimulated ATPase in sarcolemma exhibited two activation sites — one with low affinity (Km = 0.70 ± 0.2 mM; Vmax = 10.0 ± 2.2 mol Pi/mg/h) and the other with high affinity (Km = 0.16 ± 0.7 mM; Vmax = 4.6 ± 0.8 mol Pi/mg/h) for Mg2+ATP. Alamethicin at a ratio of 1:1 with the sarcolemmal protein caused a 3-fold activation of Ca2+-stimulated ATPase without affecting its sensitivity to Ca2+ or Mg2+ATP. Treatment of sarcolemma with deoxycholate or sodium dodecyl sulfate resulted in a total loss of the enzyme activity; high concentrations of alamethicin also showed a detergent-like action on the sarcolemmal vesicles. DTT at 5–10 mM concentrations caused a 4–5 fold activation of Ca2+-stimulated ATPase in sarcolemma and this effect was observed to be dependent on the concentration of Mg2+ATP. DTT increased the affinity of the enzyme to Mg2+ATP at the high affinity site and enhanced the Vmax at the low affinity site in addition to increasing the sensitivity of Ca2+-stimulated ATPase to Ca2+. DTT protected the Ca2+-stimulated ATPase against deterioration by detergents and restored the enzyme activity after treatment with N-ethylmaleimide. The mechanism of action of DTT on Ca2+-stimulated ATPase may involve the reduction of essential thiols at the active site of the enzyme or its interaction with specific DTT-dependent inhibitor protein. No changes in the sensitivity of sarcolemmal Ca2+-stimulated ATPase to orthovanadate was evident in the absence or presence of DTT and alamethicin. The results suggest the use of both DTT and alamethicin for the determination of Ca2+-stimulated ATPase activity in sarcolemmal preparations.  相似文献   

18.
The reaction of plasma membrane ATPase from yeast with Mg2+ and Mg · ATP was studied in a temperature range of 10 – 30°C. The random mechanism of activation by Mg2+ and the pseudocompetitive inhibition at higher concentrations was not altered when the temperature was varied, nor were the kinetic constants representing substrate binding. However, at low temperature, the affinity of the enzyme for Mg2+ is greatly reduced. The Arrhenius plot of log V vs. 1/T shows straight lines with an inflection point at 24°C, which disappears in the presence of detergent. Calorimetric studies of the plasma membranes show a transition point at the same temperature. From these findings we suppose that Mg2+ is bound at a regulatory site of the ATPase, which is influenced by the surrounding phospholipids.  相似文献   

19.
I. Struve  U. Lüttge 《Planta》1987,170(1):111-120
Membrane vesicles were isolated from mesophyll cells of Mesembryanthemum crystallinum in the C3 state and in the crassulacean acid metabolism (CAM) state. The distribution of ATP-hydrolysis and H+-transport activities, and the activities of hydroxypyruvate reductase and Antimycin-insensitive cytochrome-c-reductase on continuous sucrose gradients was studied. For isolations carried out routinely a discontinuous sucrose gradient (24%/37%/50%) was used. Nitrate-sensitive ATP-hydrolysis and H+-transport activities increased several-fold during the transition from C3 photosynthesis to CAM. Nitrate-sensitive ATPase showed a substrate preference for ATP with an apparent Km (MgATP2-) of 0.19–0.37 mM. In both C3 and CAM states the ATPase showed a concentration-dependent stimulation by the anions chloride and malate. However, the pH optima of the two states were different: the ATPase of C3- M. crystallinum had an optimum of pH 7.4 and that of CAM-M. crystallinum an optimum of pH 8.4. The optical probe oxonol-VI was used to demonstrate the formation of MgATP2--dependent electric-potential gradients in tonoplast vesicles.Abbreviations Bistris-Pronane 1,3-bis [tris(hydroxymethyl)-methylaminol propane - CAM Crassulacean acid metabolism - DIDS 4,4-dilsothiocyano-2,2-stilbene disulfonic acid: - DTT dithiothreitol - ER endoplasmic reticulum - Hepes 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid - HPR hydroxypyruvate reductase - IDPase inosine 5-diphosphatase - OX-VI oxonol VI - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol  相似文献   

20.
A fluorescent photoaffinity label—8-azido-1-N6-etheno-adenosine 5′-triphosphate (8-N3ε ATP)—for ATP-binding proteins has been synthesized. The effectiveness of the label is demonstrated with F1ATPase from Micrococcus luteus. 8-N3ε ATP is a substrate for the enzyme in the presence of bivalent cations. Ultraviolet irradiation of F1ATPase in the presence of the label and Mg2+ ions inhibits the enzyme irreversibly. The fluorescent label is bound preferentially to the β subunit of the enzyme. Labeling and inactivation are decreased by protection with ATP or ADP.  相似文献   

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