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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 Ca 2+ > Mg 2+ > Mn 2+= Zn 2+ > Co 2+ > Ni 2+, with all other divalent cations tested being inhibitory. Double reciprocal plots of the Ca 2+- and Mg 2+-dependent ATPase velocities as a function of substance concentration were nonlinear, suggesting the presence of multiple catalytic sites. Both MgATP 2- and CaATP 2- served as the true substrates and apparently bind to the same catalytic sites. Free ATP and Ca 2+ could inhibitit the Ca 2+- and Mg 2+-dependent ATPase. Increasing free Mg 2+ levels enhanced the affinity of the Mg 2+-dependent ATPase for MgATP 2-, while slightly inhibiting the V max values. Other divalent cation-nucleoside triphosphate complexes produced maximal enzyme velocities equal to or greater than those generated by CaATP 2- and MgATP 2-. However, the ATPase had significantly higher affinities for CaATP 2- and MgATP 2-, than for the alternative substrates. The high and low affinity components of the Ca 2+- and Mg 2+-dependent ATPase exhibited optimal V max values at pH 5 and 6, respectively. Analysis of the pH-dependence of the enzyme K m 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 F 1 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 Mg 2+. Although the equations giving the reaction rate are similar in the two cases, the calculated values of K
m
are widely different. The value obtained from the classical mechanism does not agree with K
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 Mg 2+, than by the classical mechanism. 相似文献
3.
Some aspects of the Escherichia 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 Mg 2+ 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 Mg 2+ 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 Mg 2+ ions without altering the efficiency of catalysis of ATP hydrolysis (based on the k
cat value), and by a change in the type of inhibition of ATP hydrolysis by the (ADP-Mg) − complex (without changing the K
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 Mg 2+. The free Mg 2+ concentration required for 50% inhibition of PFK activity was 0.22 millimolar. Inhibition by free Mg 2+ was independent of the MgATP 2− concentration. Inorganic phosphate (Pi) reduces the inhibition of PFK activity by Mg 2+. Free ATP (ATP 4−) also inhibits PFK activity. For free ATP the inhibition of PFK activity was dependent on the MgATP 2− concentration. Fifty percent inhibition of PFK activity requires 1.2 and 3.7 millimolar free ATP at 0.1 and 0.5 millimolar MgATP 2−, respectively. It was proposed that free ATP competes for the MgATP 2− binding site, whereas free Mg 2+ does not. Pi diminished the inhibitory effect of free ATP on PFK activity. Free ATP and Pi had substantial effects on the MgATP 2− requirement of cytosolic PFK. For half-maximum saturation of PFK activity 3 and 76 micromolar MgATP 2− 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 MgATP 2−. PFK activity was inhibited by Ca 2+. The inhibition by Ca 2+ depends upon the total Mg 2+ concentration. Fifty percent inhibition of PFK activity required 22 and 32 micromolar Ca 2+ at 0.1 and 0.2 millimolar Mg 2+, respectively. At physiological concentrations of about 0.5 millimolar free Mg 2+, Ca 2+ would have little effect on cytosolic PFK activity from spinach leaves. PFK is not absolutely specific for the nucleoside 5′-triphosphate substrate. Besides MgATP 2−, MgUTP 2−, MgCTP 2−, and MgGTP 2− 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 Mg 2+ 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 F 1F o-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 Mg 2+ 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 Mg 2+ and in the presence of Mg 2+ 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 V max rather than a change in the affinity of the enzyme for MgATP. The ATPase was inhibited by N,N-dicyclohexylcarbodiimide, diethylstilbestrol, mersalyl and KNO 3 but other inhibitors tested (azide, oligomycin, orthovanadate, K 3[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 ATP F
free ATP
- ATP T
total ATP
- BSA
bovine serum albumen
- DCCD
N,N-dicyclohexylcarbodiimide
- DES
diethylstilbestrol
- DNP
2,4-dinitrophenol
- EDAC
ethyl-3-(3-dimethylaminopropyl)carbodiimide
- K m
apparent Michaelis constant
- MgATP
complex of Mg 2+ and ATP
- Mg
F
2+
free Mg 2+
- Mg
T
2
total Mg 2+
- MES
2-( N-Morpholino)ethanesulphonic acid
- Na 2EDTA
disodium ethylenediaminetetraacetic acid
- NEM
N-ethylmaleimide
- P i
inorganic phosphate
- TCA
trichloroacetic acid
- Tris
tris(hydroxymethyl)methylamine
- V max
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 ATP 4? on adenylate cyclase. In the absence or presence of glucagon, free Mg 2+ activated adenylate cyclase by decreasing the Km for MgATP 2? without changing V. Free ATP 4? 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 Mg 2+ increased. One explanation of the effect of fluoride, consistent with the idea that free Mg 2+ activates adenylate cyclase and free ATP is not an inhibitor, is that fluoride increases the affinity of the enzyme for Mg 2+. Weak inhibition of adenylate cyclase by ATP 4? 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 NO 3− while the other contained a H +-ATPase inhibited by vanadate. We have previously proposed that these H +-ATPases are associated with tonoplast (NO 3−-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 MgATP 2−, and Mg 2+ 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 Mg 2+) 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
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 Ca 2+, whereas the Ca 2+-ATPase activity of reduced thylakoids is very low. The Mg 2+-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 Mg 2+-ATPase activity. The Mg 2+-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 Mg 2+-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 Mg 2+ 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 Ca 2+ (Ca 2+-deficient enzyme) were investigated. The basal activity of membrane-associated ATPase increased during Ca 2+ starvation, while Mg 2+-activation of the enzyme decreased and even resulted in inhibition by high Mg 2+ concentration at the late stage of the Ca 2+ starvation. The complete enzyme had low basal activity and showed a Mg 2+-activated hyperbolic reaction curve in relation to ATP concentration. Ca 2+-deficient enzyme with high basal activity showed a biphasic reaction curve and Mg 2+-activation was seen only at high ATP concentrations. Activation of membrane-associated ATPase by various cations was decreased or lost during Ca 2+ starvation. The basal ATPase activity of Ca 2+-deficient enzyme increased for various substrates including pyrophosphate, p-nitrophenyl phosphate, glucose-6 phosphate, β-glycerophosphate, AMP, ADP and ATP. Mg 2+-activation was found only for ADP and ATP in both the complete and Ca 2+-deficient enzymes, but the activation for ATP was greatly reduced by Ca 2+ starvation. The heat inactivation curves for basal and Mg 2+-activated ATPase did not differ much between the complete and Ca 2+-deficient enzyme. The delipidation of membrane-associated enzyme by acetone affected the protein content and the basal activity slightly, but inhibited the Mg 2+-activated ATPase activity clearly with somewhat different behaviour between the complete and Ca 2+-deficient enzyme. 相似文献
12.
The plasma membrane Ca 2+ ATPase catalyzed the hydrolysis of ATP in the presence of millimolar concentrations of EGTA and no added Ca 2+ at a rate near 1.5% of that attained at saturating concentrations of Ca 2+. 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 Ca 2+-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 Ca 2+-independent ATP hydrolysis reflects the well known phosphatase activity which is maximal in the absence of Ca 2+ and is catalyzed by E 2-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 Mg 2+ 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-bisphosphate 4-)(Mg 2+) complex has been identified as the substrate of the enzyme. Therefore, changes of pH and Mg 2+ concentrations have an immediate effect on the activity of FBPase by shifting the pH and Mg 2+ dependent equilibrium concentration of the substrate. In addition, changes of pH and Mg 2+ concentration in the assay medium have a delayed effect on FBPase activity. A correlation of the activities observed using different pH and Mg 2+ concentrations indicates, that the effect is not a consequence of the pH and Mg 2+ concentration as such, but is caused by a shift in the equilibrium concentration of a hypothetical inhibitor fructose-1,6-bisphosphate 3- (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 Mg 2+ 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.
(1) Light-activated ‘dark’ ATPase in Rhodospirillum rubrum chromatophores is inhibited by preincubation with ADP or ATP (in the absence of Mg 2+). 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 Mg 2+ 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 Mg 2+ (see below), MgATP-induced release of tightly bound ADP or ATP does not require a transmembrane . 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 Mg 2+. In addition, MgATP-dependent reversal of ADP inhibition in the dark was inhibited by Mg 2+ 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 Mg 2+ 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 Mg 2
+ and Ca 2+ 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 [V MgATP and V CaATP: 13±1 and 10±1 nmol s –1 ATP hydrolyzed (μmol BChl) –1; K MgATP and K CaATP: 0.22±0.06 and 0.20±0.05 m m]. 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
K I coincident with K CaATP 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.
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 Mg 2+-dependent ATPase in the presence of ATP. Mathematical and graphical analysis of the data yielded a quantitative relationship between magnesium-induced stimulation of the Mg 2+-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 Ca 2+-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 Ca 2+-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 Mg 2+ATP. Alamethicin at a ratio of 1:1 with the sarcolemmal protein caused a 3-fold activation of Ca 2+-stimulated ATPase without affecting its sensitivity to Ca 2+ or Mg 2+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 Ca 2+-stimulated ATPase in sarcolemma and this effect was observed to be dependent on the concentration of Mg 2+ATP. DTT increased the affinity of the enzyme to Mg 2+ATP at the high affinity site and enhanced the Vmax at the low affinity site in addition to increasing the sensitivity of Ca 2+-stimulated ATPase to Ca 2+. DTT protected the Ca 2+-stimulated ATPase against deterioration by detergents and restored the enzyme activity after treatment with N-ethylmaleimide. The mechanism of action of DTT on Ca 2+-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 Ca 2+-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 Ca 2+-stimulated ATPase activity in sarcolemmal preparations. 相似文献
18.
The reaction of plasma membrane ATPase from yeast with Mg 2+ and Mg · ATP was studied in a temperature range of 10 – 30°C. The random mechanism of activation by Mg 2+ 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 Mg 2+ 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 Mg 2+ is bound at a regulatory site of the ATPase, which is influenced by the surrounding phospholipids. 相似文献
19.
Membrane vesicles were isolated from mesophyll cells of Mesembryanthemum crystallinum in the C 3 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 C 3 photosynthesis to CAM. Nitrate-sensitive ATPase showed a substrate preference for ATP with an apparent K m (MgATP 2-) of 0.19–0.37 mM. In both C 3 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 C 3-
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 MgATP 2--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-N 3ε ATP)—for ATP-binding proteins has been synthesized. The effectiveness of the label is demonstrated with F 1ATPase from Micrococcus luteus. 8-N 3ε ATP is a substrate for the enzyme in the presence of bivalent cations. Ultraviolet irradiation of F 1ATPase in the presence of the label and Mg 2+ 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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