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1.
Phosphoenolpyruvate carboxylase (PEPC) and pyruvate orthophosphate dikinase (PPDK) cold inactivation was studied in leaf extracts from Atriplex halimus L. Both enzyme activities gradually reduced as the temperature and the total soluble protein decreased. Mg2+ at a concentration of 10 mM stabilized PEPC and PPDK activities against cold inactivation. At low Mg2+ concentration (4 mM), PEPC was strongly protected by phosphoenolpyruvate, glucose-6-phosphate, and, partially, byL-malate, while PPDK was protected by PEP, but not by its substrate, pyruvate. High concentrations of compatible solutes (glycerol, betaine, proline, sorbitol and trehalose) proved to be good protectants for both enzyme activities against cold inactivation. When illuminated leaves were exposed to low temperature, PPDK was partially inactivated, while the activity of PEPC was not altered. 相似文献
2.
Pyruvate dehydrogenase complex activity from spinach leaf mitochondria was inhibited up to 90% within 2 min of incubation with 1 mm ATP at 27 °C. The inhibition was time, temperature and ATP concentration dependent. The inhibition was partially prevented with 3.0 mm dichloroacetate, a known inhibitor of mammalian pyruvate dehydrogenase kinases. Optimum pH for ATP-dependent inactivation was between 8.0 and 9.0 The inactivated complex was reactivated with 10 to 20 mm MgCl2. Complete reactivation occurs within 10 min after MgCl2 addition. Reactivation was inhibited by fluoride, a known inhibitor of mammalian pyruvate dehydrogenase phosphatase. Optimum pH for Mg2+-dependent reactivation was 8.0. It is concluded that the inactivation and reactivation process of pyruvate dehydrogenase complex from spinach leaf mitochondria is due to phosphorylation and dephosphorylation. 相似文献
3.
Submission of chloroplasts to alkaline pH, in the range pH 7.5–9.5, leads to changes in their oxygen-evolving capacities. These changes are enhanced by the addition of divalent cations and also monovalent cations at high concentrations. (1) Dark incubation of chloroplasts at pH ? 9 gives rise to a time-dependent inactivation of electron transport from water to 2,6-dichlorophenolindophenol measured at neutral pH. The rate of inactivation is increased by adding cations. (2) The variable fluorescence is decreased with a dependence on incubation time and concentration of cations similar to that of the Hill reaction. Addition of the electron donor NH2OH removes most of the fluorescence quenching, (3) EPR measurements indicate that the inactivations are accompanied by loss of Mn2+ and the appearance of signal II fast. (4) At lower pH (7.5) the oscillations of oxygen evolved per flash during a sequence of flashes show an increase in damping when 20 mM MgCl2 is present instead of 100 mM KCI. These changes are not seen at pH 6. (5) None of these Mg2+-induced modifications are prevented by glutaraldehyde fixation. We conclude that the effects of alkaline pH and MgCl2 do not involve major protein structural changes, and that both act on the manganese-containing protein of the oxygen-evolving site. 相似文献
4.
Effects of Magnesium on Intact Chloroplasts: I. EVIDENCE FOR ACTIVATION OF (SODIUM) POTASSIUM/PROTON EXCHANGE ACROSS THE CHLOROPLAST ENVELOPE
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Exogenous Mg2+ (2 millimolar) altered the stromal pH of intact spinach chloroplasts. Without added KCl in the medium, Mg2+ decreased the stromal pH in the light by approximately 0.3 pH unit. External KCl (25 millimolar) largely prevented the acidification caused by Mg2+. Effects on the stromal pH were not caused by changes in H+ pumping across the thylakoid membrane because Mg2+ had no effect on the light-induced quenching of atebrin fluorescence by intact chloroplasts. However, Mg2+ affected H+ fluxes across the envelope. Addition of Mg2+ to intact chloroplasts in the dark caused a significant acidification of the medium that was dependent on the presence of K+. 相似文献
5.
Isolation and Characterization of Two Enzymes Capable of Hydrolyzing Fructose-1,6-Bisphosphatase from the Lichen Peltigera rufescens 总被引:1,自引:0,他引:1
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Two enzymes capable of hydrolyzing fructose-1,6-bisphosphate (FBP) have been isolated from the foliose lichen Peltigera rufescens (Weis) Mudd. These enzymes can be separated using Sephadex G-100 and DEAE Sephacel chromatography. One enzyme has a pH optimum of 6.5, and a substrate affinity of 228 micromolar FBP. This enzyme does not require MgCl2 for activity, and is inhibited by AMP. The second enzyme has a pH optimum of 9.0, with no activity below pH 7.5. This enzyme responds sigmoidally to Mg2+, with half-saturation concentration of 2.0 millimolar MgCl2, and demonstrates hyperbolic kinetics for FBP (Km = 39 micromolar). This enzyme is activated by 20 millimolar dithiothreitol, is inhibited by AMP, but is not affected by fructose-2-6-bisphosphate. It is hypothesized that the latter enzyme is involved in the photosynthetic process, while the former enzyme is a nonspecific acid phosphatase. 相似文献
6.
Conditions for extraction and assay of ribulose-1,5-bisphophate carboxylase present in an in vivo active form (initial activity) and an inactive form able to be activated by Mg2+ and CO2 (total activity) were examined in leaves of soybean, Glycine max (L.) Merr. cv Will. Total activity was highest after extracts had preincubated in NaHCO3 (5 millimolar saturating) and Mg2+ (5 millimolar optimal) for 5 minutes at 25°C or 30 minutes at 0°C before assay. Initial activity was about 70% of total activity. Kact (Mg2+) and Kact (CO2) were approximately 0.3 millimolar and 36 micromolar, respectively. The carry-over of endogenous Mg2+ in the leaf extract was sufficient to support considerable catalytic activity. While Mg2+ was essential for both activation and catalysis, Mg2+ levels greater than 5 millimolar were increasingly inhibitory of catalysis. Similar inhibition by high Mg2+ was also observed in filtered, centrifuged, or desalted extracts and partially purified enzyme. Activities did not change upon storage of leaves for up to 4 hours in ice water or liquid nitrogen before homogenization, but were about 20% higher in the latter. Activities were also stable for up to 2 hours in leaf extracts stored at 0°C. Initial activity quickly deactivated at 25°C in the absence of high CO2. Total activity slowly declined irreversibly upon storage of leaf homogenate at 25°C. 相似文献
7.
Huber SC 《Plant physiology》1978,62(3):321-325
Magnesium was most inhibitory to photosynthetic reactions by intact chloroplasts when the magnesium was added in the dark before illumination. Two millimolar MgCl2, added in the dark, inhibited CO2-dependent O2 evolution by Hordeum vulgare L. and Spinacia oleracea L. (C3 plants) chloroplasts 70 to 100% and inhibited (pyruvate + oxaloacetate)-dependent O2 evolution by Digitaria sanguinalis L. (C4 plant) mesophyll chloroplasts from 80 to 100%. When Mg2+ was added in the light, O2 evolution was reduced only slightly. O2 evolution in the presence of phosphoglycerate was less sensitive to Mg2+ inhibition than was CO2-dependent O2 evolution.
Magnesium prevented the light activation of several photosynthetic enzymes. Two millimolar Mg2+ blocked the light activation of NADP-malate dehydrogenase in D. sanguinalis mesophyll chloroplasts, and the light activation of phosphoribulokinase, NADP-linked glyceraldehyde-3-phosphate dehydrogenase, and fructose 1,6-diphosphatase in barley chloroplasts. The results suggest that Mg2+ inhibits chloroplast photosynthesis by preventing the light activation of certain enzymes.
相似文献8.
Activation of Ribulosebisphosphate Carboxylase/Oxygenase at Physiological CO(2) and Ribulosebisphosphate Concentrations by Rubisco Activase 总被引:29,自引:20,他引:9
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The enzyme-catalyzed activation of ribulosebisphosphate carboxylase/oxygenase (rubisco) was investigated in an illuminated reconstituted system containing thylakoid membranes, rubisco, ribulosebisphosphate (RuBP), MgCl2, carbonic anhydrase, catalase, the artificial electron acceptor pyocyanine, and partially purified rubisco activase. Optimal conditions for light-induced rubisco activation were found to include 100 micrograms per milliliter rubisco, 300 micrograms per milliliter rubisco activase, 3 millimolar RuBP, and 6 millimolar free Mg2+ at pH 8.2. The half-time for rubisco activation was 2 minutes, and was 4 minutes for rubisco deactivation. The rate of rubisco deactivation was identical in the presence and absence of activase. The Kact(CO2) of rubisco activation in the reconstituted system was 4 micromolar CO2, compared to a Kact(CO2) of 25 to 30 micromolar CO2 for the previously reported spontaneous CO2/Mg2+ activation mechanism. The activation process characterized here explains the high degree of rubisco activation at the physiological concentrations of 10 micromolar CO2 and 2 to 4 millimolar RuBP found in intact leaves, conditions which lead to almost complete deactivation of rubisco in vitro. 相似文献
9.
Effects of Magnesium on Intact Chloroplasts : II. CATION SPECIFICITY AND INVOLVEMENT OF THE ENVELOPE ATPase IN (SODIUM) POTASSIUM/PROTON EXCHANGE ACROSS THE ENVELOPE 总被引:23,自引:22,他引:1
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Addition of exogenous Mg2+ (2 millimolar) to illuminated intact spinach (Spinacia oleracea L.) chloroplasts caused acidification of the stroma and a 20% decrease in stromal K+. Addition of K+ (10-50 millimolar) reversed both stromal acidification and K+ efflux from the chloroplast caused by Mg2+. These data suggested that Mg2+ induced reversible H+/K+ fluxes across the chloroplast envelope. Ca2+ and Mn2+ (2 millimolar) were as effective as 4 millimolar Mg2+ in causing K+ efflux from chloroplasts and inhibition of O2 evolution. In contrast, 10 millimolar Ba2+ induced only a small amount of inhibition. The lack of strong inhibition by Ba2+ indicated that the effects of divalent cations such as Mg2+ cannot be attributed to generalized electrostatic interactions of the cation with the chloroplast envelope. With the chloroplasts used in this study, stromal acidification caused by 2 millimolar Mg2+ was small (0.07 to 0.15 pH units), but sufficient to account for the inhibition of O2 evolution (43%) induced by Mg2+. 相似文献
10.
Characterization of a partially purified adenosine triphosphatase from a corn root plasma membrane fraction 总被引:19,自引:18,他引:1
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The (K+,Mg2+)-ATPase was partially purified from a plasma membrane fraction from corn roots (WF9 × Mol7) and stored in liquid N2 without loss of activity. Specific activity was increased 4-fold over that of the plasma membrane fraction. ATPase activity resembled that of the plasma membrane fraction with certain alterations in cation sensitivity. The enzyme required a divalent cation for activity (Co2+ > Mg2+ > Mn2+ > Zn2+ > Ca2+) when assayed at 3 millimolar ATP and 3 millimolar divalent cation at pH 6.3. When assayed in the presence of 3 millimolar Mg2+, the enzyme was further activated by monovalent cations (K+, NH4+, Rb+ Na+, Cs+, Li+). The pH optima were 6.5 and 6.3 in the absence and presence of 50 millimolar KCl, respectively. The enzyme showed simple Michaelis-Menten kinetics for the substrate ATP-Mg, with a Km of 1.3 millimolar in the absence and 0.7 millimolar in the presence of 50 millimolar KCl. Stimulation by K+ approached simple Michaelis-Menten kinetics, with a Km of approximately 4 millimolar KCl. ATPase activity was inhibited by sodium orthovanadate. Half-maximal inhibition was at 150 and 35 micromolar in the absence and presence of 50 millimolar KCl. The enzyme required the substrate ATP. The rate of hydrolysis of other substrates, except UDP, IDP, and GDP, was less than 20% of ATP hydrolysis. Nucleoside diphosphatase activity was less than 30% of ATPase activity, was not inhibited by vanadate, was not stimulated by K+, and preferred Mn2+ to Mg2+. The results demonstrate that the (K+,Mg2+)-ATPase can be clearly distinguished from nonspecific phosphohydrolase and nucleoside diphosphatase activities of plasma membrane fractions prepared from corn roots. 相似文献
11.
An essential arginyl residue in the tonoplast pyrophosphatase from etiolated mung bean seedlings 总被引:3,自引:3,他引:0
Tonoplast membrane of etiolated mung bean (Vinga radiata. L.) seedlings contained H+-translocating pyrophosphatase (PPase). Modification of tonoplast vesicles and partially purified PPase from etiolated mung bean seedlings with arginine-specific reagents, phenylglyoxal (PGO) and 2,3-butanedione (BD), resulted in a marked decline in H+-translocating PPase activity. The half-maximal inhibition was brought about by 20 millimolar PGO and 50 millimolar BD for membrane bound and 1.5 millimolar PGO and 5.0 millimolar BD for soluble PPase, respectively. The substrate, Mg2+-pyrophosphate, provided partial protection against inactivation by these reagents. Loss of activity of partially purified PPase followed pseudo-first order kinetics. The double logarithm plots of pseudo-first order rate constant versus reagent concentrations gave slopes of 0.88 (PGO) and 0.90 (BD), respectively, suggesting that the inactivation may possibly result from reaction of at least one arginyl residue at the active site of H+-translocating PPase. 相似文献
12.
J. Ashot Kozak 《Biophysical journal》2003,84(2):922-927
TRPM7 channels are nonselective cation channels that possess a functional α-kinase domain. It has been proposed that heterologously expressed TRPM7 channels are activated (Runnels et al., 2001) or inhibited (Nadler et al., 2001) by dialyzing the cell with millimolar levels of ATP. The endogenous correlate of TRPM7 has been identified in T-lymphocytes and RBL (rat basophilic leukemia) cells and named MagNuM (for Mg2+-nucleotide-inhibited metal) or MIC (for Mg2+-inhibited cation). Here, we report that internal Mg2+ rather than MgATP inhibits this current. Cytoplasmic MgATP, supplied by dialysis at millimolar concentrations, effectively inhibits only when a weak Mg2+ chelator is present in the pipette solution. Thus, MgATP acts as a source of Mg2+ rather than a source of ATP. Using an externally accessible site within the pore of the MIC channel itself as a bioassay, we show that equimolar MgCl2 and MgATP solutions contain similar amounts of free Mg2+, explaining the fact that numeric values of Mg2+ and MgATP concentrations necessary for complete inhibition are the same. Furthermore, we demonstrate that Mg2+ is not unique in its inhibitory action, as Ba2+, Sr2+, Zn2+, and Mn2+ can substitute for Mg2+, causing complete inhibition. We conclude that MIC current inhibition occurs simply by divalent cations. 相似文献
13.
Disruption of the coat of coated vesicles is accompanied by the release of clathrin and other proteins in soluble form. The ability of solubilized coated vesicle proteins to reassemble into empty coats is influenced by Mg2+, Tris ion concentration, pH, and ionic strength. The proteins solubilized by 2 M urea spontaneously reassemble into empty coats following dialysis into isolation buffer (0.1 M MES–1 mM EGTA–1 mM MgCl2–0.02% NaN3, pH 6.8). Such reassembled coats have sedimentation properties similar to untreated coated vesicles. Clathrin is the predominant protein of reassembled coats; most of the other proteins present in native coated vesicles are absent. We have found that Mg2+ is important in the coat assembly reaction. At pH 8 in 0.01 M or 0.1 M Tris, coats dissociate; however, 10 mM MgCl2 prevents dissociation. If the coats are first dissociated at pH 8 and then the MgCl2 raised to 10 mM, reassembly occurs. These results suggest that Mg2+ stabilizes the coat lattice and promotes reassembly. This hypothesis is supported by our observations that increasing Mg2+ (10 μM–10 mM) increases reassembly whereas chelation of Mg2+ by (EGTA) inhibits reassembly. Coats reassembled in low-Tris (0.01 M, pH 8) supernatants containing 10 mM MgCl2 do not sediment, but upon dialysis into isolation buffer (pH 6.8), these coats become sedimentable. Nonsedimentable coats are noted also either when partially purified clathrin (peak I from Sepharose CL4B columns) is dialyzed into low-ionic-strength buffer or when peaks I and II are dialyzed into isolation buffer. Such nonsedimentable coats may represent intermediates in the assembly reaction which have normal morphology but lack some of the physical properties of native coats. We present a model suggesting that tightly intertwined antiparallel clathrin dimers form the edges of the coat lattice. 相似文献
14.
Isolation and Characterization of Phosphoenolpyruvate Phosphatase from Germinating Mung Beans (Vigna radiata) 总被引:15,自引:7,他引:8
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A phosphoenolpyruvate (PEP) phosphatase was purified to homogeneity from germinating mung beans (Vigna radiata). It was found to be a tetrameric protein (molecular mass 240,000 daltons) made up of apparently identical subunits (subunit molecular mass 60,000 daltons). It was free from bound nucleotides. It did not show pyruvate kinase activity. The enzyme showed high specificity for PEP. Pyrophosphate and some esters (nucleoside di- and triphosphates) were hydrolyzed slowly and phosphoric acid monoesters were not hydrolyzed. The enzyme showed maximum activity at pH 8.5. At this pH, the Km of PEP was 0.14 millimolar and the Vmax was equal to 1.05 micromoles pyruvate formed per minute per milligram enzyme protein. Dialysis of the enzyme against 10 millimolar triethanolamine buffer (pH 6.5), led to loss of the catalytic activity, which was restored on addition of Mg2+ ions (Km = 0.12 millimolar). Other divalent metal ions inhibited the Mg2+ -activated enzyme. PEP-phosphatase was inhibited by ATP and several other metabolites. 相似文献
15.
A procedure was developed to isolate a membrane fraction of rat skeletal muscle which contains a highly active Mg2+-ATPase (5–25 μmol Pi/mg min). The rate of ATP hydrolysis by the Mg2+-ATPase was nonlinear but decayed exponentially (first-order rate constant ≥0.2 s?1 at 37°C). The rapid decline in the ATPase activity depended on the presence of ATP or its nonhydrolyzable analog 5′-adenylyl imidodiphosphate (AdoPP[NH]P). Once inactivated, removal of ATP from the medium did not immediately restore the original activity. ATP- or AdoPP[NH]P-dependent inactivation could be blocked by concanavalin A, wheat germ agglutinin or rabbit antiserum against the membrane. Additions of these proteins after ATP addition prevented further inactivation but did not restore the original activity. Low concentrations of ionic and nonionic detergents increased the rate of ATP-dependent inactivation. Higher concentrations of detergents, which solubilize the membrane completely, inactivated the Mg2+-ATPase. Cross-linking the membrane components with glutaraldehyde prevented ATP-dependent inactivation and decreased the sensitivity of the Mg2+-ATPase to detergents. It is proposed that the regulation of the Mg2+-ATPase by ATP requires the mobility of proteins within the membrane. Cross-linking the membrane proteins with lectins, antiserum or glutaraldehyde prevents inactivation; increasing the mobility with detergents accelerates ATP-dependent inactivation. 相似文献
16.
Isolation of dihydroxyacetone phosphate reductase from dunaliella chloroplasts and comparison with isozymes from spinach leaves
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A dihydroxyacetone phosphate (DHAP) reductase has been isolated in 50% yield from Dunaliella tertiolecta by rapid chromatography on diethylaminoethyl cellulose. The activity was located in the chloroplasts. The enzyme was cold labile, but if stored with 2 molar glycerol, most of the activity was restored at 30°C after 20 minutes. The spinach (Spinacia oleracea L.) reductase isoforms were not activated by heat treatment. Whereas the spinach chloroplast DHAP reductase isoform was stimulated by leaf thioredoxin, the enzyme from Dunaliella was stimulated by reduced Escherichia coli thioredoxin. The reductase from Dunaliella was insensitive to surfactants, whereas the higher plant reductases were completely inhibited by traces of detergents. The partially purified, cold-inactivated reductase from Dunaliella was reactivated and stimulated by 25 millimolar Mg2+ or by 250 millimolar salts, such as NaCl or KCl, which inhibited the spinach chloroplast enzyme. Phosphate at 3 to 10 millimolar severely inhibited the algal enzyme, whereas phosphate stimulated the isoform in spinach chloroplasts. Phosphate inhibition of the algal reductase was partially reversed by the addition of NaCl or MgCl2 and totally by both. In the presence of 10 millimolar phosphate, 25 millimolar MgCl2, and 100 millimolar NaCl, reduced thioredoxin causes a further twofold stimulation of the algal enzyme. The Dunaliella reductase utilized either NADH or NADPH with the same pH maximum at about 7.0. The apparent Km (NADH) was 74 micromolar and Km (NADPH) was 81 micromolar. Apparent Vmax was 1100 μmoles DHAP reduced per hour per milligram chlorophyll for NADH, but due to NADH inhibition highest measured values were 350 to 400. The DHAP reductase from spinach chloroplasts exhibited little activity with NADPH above pH 7.0. Thus, the spinach chloroplast enzyme appears to use NADH in vivo, whereas the chloroplast enzyme from Dunaliella or the cytosolic isozyme from spinach may utilize either nucleotide. 相似文献
17.
A second fructokinase (EC 2.7.1.4) was obtained from pea seed (Pisum sativum L. var. Progress No. 9) extracts. The enzyme, termed fructokinase (fraction III), was specific for fructose and had little activity with glucose. With fructose concentrations above 0.25 millimolar, there was strong substrate inhibition at the optimum pH (8.0) and also at pH 6.6. The apparent Km values at pH 8.0 for fructose and glucose were 0.06 millimolar and 0.14 millimolar, respectively. The apparent Km for Mg adenosine 5′-triphosphate (MgATP) was 0.06 millimolar and excess MgATP was inhibitory. Mg2+ was essential for activity but the enzyme was inhibited by excess Mg2+ or ATP. Mg adenosine 5′-pyrophosphate was also inhibitory. Activity was stimulated by the addition of monovalent cations: of those tested K+, Rb+, and NH4+ were the most effective. The possible role of fructokinase (fraction III) is discussed. 相似文献
18.
The regulatory properties of NADH-dependent nitrate reductase (NR) in desalted root extracts from hydroponically grown cucumber
(Cucumis sativus L.) seedlings were examined. The lowest activity of NR was detected in extracts incubated with Mg2+ and ATP. An inhibitory effect of Mg-ATP was cancelled in the presence of staurosporine (the protein kinase inhibitor) and
completely reversed after addition of ethylenediaminetetraacetate (EDTA) as well as AMP into reaction mixture. Reactivation
of enzyme due to AMP presence, contrary to the chelator-dependent NR activation, was sensitive to microcystin LR (the protein
phosphatase inhibitor). Above results indicated that the nitrate reductase in cucumber roots was regulated through reversible
phosphorylation of enzyme protein. A drop in the activity of NR was also observed after incubation of enzyme at low pH. At
low pH, the presence of ATP alone in the incubation medium was sufficient to inactivate NR, indicating that H+ can substitute the Mg2+ in formation of an inactive complex of enzyme. ATP-dependent inactivation of NR at low pH was prevented by staurosporine
and reversed by AMP. However, AMP action was not altered by microcystin LR suggesting that in low pH the nucleotide induced
reactivation of NR is not limited to the protein phosphorylation. 相似文献
19.
Nitrate reductase in spinach (Spinacia oleracea L.) leaves was rapidly inactivated in the dark and reactivated by light, whereas in pea (Pisum sativum L.), roots, hyperoxic conditions caused inactivation, and anoxia caused reactivation. Reactivation in vivo, both in leaves and roots, was prohibited by high concentrations (10–30 M) of the serine/threonine-protein phosphatase inhibitors okadaic acid or calyculin, consistent with the notion that protein dephosphorylation catalyzed by type-1 or type-2A phosphatases was the mechanism for the reactivation of NADH-nitrate reductase (NR). Following inactivation of leaf NR in vivo, spontaneous reactivation in vitro (in desalted extracts) was slow, but was drastically accelerated by removal of Mg2+ with excess ethylenediaminetetraacetic acid (EDTA), or by desalting in a buffer devoid of Mg2+. Subsequent addition of either Mg2+, Mn2+ or Ca2+ inhibited the activation of NR in vitro. Reactivation of NR (at pH 7.5) in vitro in the presence of Mg2+ was also accelerated by millimolar concentrations of AMP or other nucleoside monophosphates. The EDTA-mediated reactivation in desalted crude extracts was completely prevented by protein-phosphatase inhibitors whereas the AMP-mediated reaction was largely unaffected by these toxins. The Mg2+-response profile of the AMP-accelerated reactivation suggested that okadaic acid, calyculin and microcystin-LR were rather ineffective inhibitors in the presence of divalent cations. However, with partially purified enzyme preparations (5–15% polyethyleneglycol fraction) the AMPmediated reactivation was also inhibited (65–80%) by microcystin-LR. Thus, the dephosphorylation (activation) of NR in vitro is inhibited by divalent cations, and protein phosphatases of the PP1 or PP2A type are involved in both the EDTA and AMP-stimulated reactions. Evidence was also obtained that divalent cations may regulate NR-protein phosphatase activity in vivo. When spinach leaf slices were incubated in Mg2+ -and Ca2+-free buffer solutions in the dark, extracted NR was inactive. After addition of the Ca2+ /Mg2+-ionophore A 23187 plus EDTA to the leaf slices, NR was activated in the dark. It was again inactivated upon addition of divalent cations (Mg2+ or Ca2+). It is tentatively suggested that Mg2+ fulfills several roles in the regulatory system of NR: it is required for active NR-protein kinase, it inactivates the protein phosphatase and is, at the same time, necessary to keep phospho-NR in the inactive state. The EDTA- and AMP-mediated reactivation of NR in vitro had different pH optima, suggesting that two different protein phosphatases may be involved. At pH 6.5, the activation of NR was relatively slow and the addition or removal of Mg2+ had no effect. However, 5-AMP was a potent activator of the reaction with an apparent K
m of 0.5 mM. There was also considerable specificity for 5AMP relative to 3- or 2-AMP or other nucleoside monophoposphates. We conclude that, depending upon conditions, the signals triggering NR modulation in vivo could be either metabolic (e.g. 5-AMP) or physical (e.g. cytosolic [Mg2+]) in nature.Abbreviations DTT
dithiothreitol
- Mops
3-(N-morpholino)propanesulfonic acid
- NR
NADH-nitrate reductase
- NRA
nitrate-reductase activity
- PP
protein phosphatase
This paper is dedicated to Prof. O.K. Volk on the occasion of his 90th birthdayThe skilled technical assistance of Elke Brendle-Behnisch is gratefully acknowledged. The investigations were cooperatively supported by the Deutsche Forschungsgemeinschaft (SFB 251), the U.S. Department of Agriculture, Agricultural Research Services, Raleigh, NC. This work was also supported in part by a grant from the U.S. Department of Energy (Grant DE-A I05-91 ER 20031 to S.C.H.). 相似文献
20.
《生物化学与生物物理学报:生物膜》1990,1030(1):152-156
The (Ca2+ + Mg2+-ATPase of sarcoplasmic reticulum catalyzes the hydrolysis of acetyl phosphate in the presence of Mg2+ and EGTA and is stimulated by Ca2+. The Mg2+-dependent hydrolysis of acetyl phosphate measured in the presence of 6 mM acetyl phosphate, 5mM MgCl2, and 2 mM EGTA is increased 2-fold by 20% dimethyl sulfoxide. This activity is further stimulated 1.6-fold by the addition of 30 mM KCl. In this condition addition of Ca2+ causes no further increase in the rate of hydrolysis and Ca2+ uptake is reduced to a low level. In leaky vesicles, hydrolysis continues to be back-inhibited by Ca2+ in the millimolar range. Unlike ATP, acetyl phosphate does not inhibit phosphorylation by Pi unless dimethyl sulfoxide is present. The presence of dimethyl sulfoxide also makes it possible to detect Pi inhibition of the Mg2+-dependent acetyl phosphate hydrolysis. These results suggest that dimethyl sulfoxide stabilizes a Pi-reactive form of the enzyme in a conformation that exhibits comparable affinities for acetyl phosphate and Pi. In this conformation the enzyme is transformed from a Ca2+- and Mg2+-dependent ATPase into a (K+ + Mg2+)-ATPase. 相似文献