Abbreviations: Rib-5-P, ribose 5-phosphate; Ribul-5-P, ribulose 5-phosphate; Ribul-1,5-P2, ribulose 1,5-diphosphate; HEPES, N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid; MES, 2-(N-morpholino)ethanesulfonic acid 相似文献
The Mg2+-dependent or the (Mg2+ + Na+)-dependent phosphorylation of ADP by the phospho-protein (the exchange reaction) is reversibly inhibited when the microsomes are incubated with hydroxylamine.
The Na+-induced increment of 32P-labelling of microsomes previously incubated with [λ-32P]ATP is completely eliminated by hydroxylamine, but the Mg2+-dependent 32P-labelling of such microsomes is unaffected by hydroxylamine.
It is concluded that the phospho-enzyme formed during the Mg2+-dependent hydrolysis does not contribute to the Mg2+-dependent exchange reaction. Instead, the phosphoenzyme formed during the (Na+ + K+)-stimulated hydrolysis is apparently the only substance which phosphorylates ADP in the exchange reaction, even in the absence of Na+ and/or K+.
The hydroxylamine-sensitive nature of the sodium form of the phospho-enzyme in the (Na+ + K+)-stimulated ATPase sequence is consistent with the existence of an enzyme-acyl-phosphate bond of high internal energy with respect to that of ADP.
On the other hand, the hydroxylamine-resistant nature of the phospho-enzyme in the Mg2+-ATPase sequence suggests the existence of a non-acyl type of enzyme phosphate bond with low internal energy relative to that of ADP. 相似文献
1. 1. The effect of the Mg2+ concentration on the CO2 fixation activity in situ in isolated and intact spinach chloroplasts upon suspension in hypotonic medium was examined. CO2 fixation in the dark was activated 25–100 fold by 20 mM Mg2+ in the presence of added ATP plus either ribulose 5-phosphate or ribose 5-phosphate. 20 mM Mg2+-stimulated fixation only 2–3 fold in the presence of the substrate of fixation, ribulose 1,5-diphosphate. The highest Mg2+-stimulated rate of fixation in the dark observed with chloroplasts was 480 μmoles CO2 fixed per mg chlorophyll per h.
2. 2. The concentration of bicarbonate at half of the maximal velocity (apparent Km) during the Mg2+-stimulated fixation of CO2 was 0.4 mM in the presence of ATP plus ribose 5-phosphate and 0.6 mM with ribulose 1,5-diphosphate.
3. 3. Dithioerythritol or light enhanced Mg2+-stimulated CO2 fixation 1–3 fold in the presence of ATP plus ribose 5-phosphate but not ribulose 1,5-diphosphate.
4. 4. These results indicate that Mg2+ fluxes in the stroma of the chloroplast could control the activity of the phosphoribulokinase with a lesser effect on the ribulosediphosphate carboxylase. An increase in Mg2+ of 6–10 mM in the stroma region of the chloroplast would be enough to activate CO2 fixation during photosynthesis.
1. 1. (Mg2+ + Ca2+) ATPases of microsomal and synaptic membrane preparations from immature and adult rat brain were activated by calcium (0.1–10 μM), maximal activation was found at 3 μM. The increase in (Mg2+ + Ca2+) ATPase seen during development was greatest in the synaptic membrane preparations.
2. 2. At 37°C both Na+ or K+ at concentrations higher than 30 mM inhibited the microsomal Mg2+ ATPase, but the (Mg2+ + Ca2+) ATPase was stimulated by both Na+ and K+. Synaptic membrane Mg2+ ATPase was inhibited by concentrations higher than 100 mM K+; Na+ however stimulated this enzyme at all concentrations. Much of this Na+ stimulated activity was ouabain sensitive. Synaptic membrane (Mg2+ + Ca2+) ATPase was stimulated by Na+ or K+, this stimulation follows approximate saturation kinetics with an apparent Km of 18.8 mM Na+ or K+.
3. 3. Arrhenius plots of microsomal (Mg2+ + Ca2+) ATPase were curvilinear, but two intersecting lines with a break at 20°C could be fitted. The calculated energies of activation from these lines were very similar in immature and adult preparations. The synaptic membrane preparation (adult) also gave a curvilinear plot; but two intersecting lines with a break at 25°C could be fitted to the data. These lines had slopes of 21 and 28 Kcal mole−1 above and below the break, respectively. The immature preparation when made using EDTA gave a Arrhenius plot of very similar form to the adult preparation. Without EDTA however the Arrhenius plot was complex with a plateau at 25–32°C. Pretreatment with EDTA activated the synaptic membrane (Mg2+ + Ca2+) ATPase from both immature and adult brain.
Author Keywords: Brain; ATPase; temperature; development; synaptic membranes 相似文献
The Bers’ Ca2+ macroelectrode method [Bers, D. M., 1982 A simple method for the determination of free [Ca] in Ca-EGTA solutions Am. J. Physiol. 242, C404–C408] cannot be used with Mg2+-macroelectrodes and is partly arbitrary since the linear part of the Scatchard plot is judged subjectively. Iterative methods have therefore been introduced. Iteration based on the Bers’ method or the lumped interference in the Nicolsky–Eisenman equation also failed with Mg2+ macroelectrodes. The Oiki et al., method [Oiki, S., Yomamoto, T., Okada, Y., 1994. Apparent stability constants and purity of Ca-chelating agents evaluated using Ca-sensitive electrodes by the double-log optimization method Cell Calcium 15, 209–46.] cannot be applied to Mg2+ macroelectrodes. The pH titration method of Moisescu and Pusch (Pflügers, Arch., 355, R122, 1975) predicted EGTA purity and Ca2+ contamination, but Kapp values for EGTA were approximate. It cannot be applied to Mg2+ binding. The partition method [Godt, R.E., 1974. Calcium-activated tension of skinned muscle fibres of the frog. Dependence on magnesium adenosine triphosphate concentration J. Gen. Physiol. 63, 722–739.] only approximately estimated the Kapp. Calibration, maintaining contaminating [Ca2+]/[Mg2+] at <1 μmol l−1, and setting standards by dilution, is the ultimate check of calculated ionised concentrations, although technically difficult. The macroelectrode method of Lüthi et al. [1997. Calibration of Mg2+-selective macromolecules down to 1 μmol l−1 in intracellular and Ca+- containing extracellular solutions. Exp. Physiol. 82, 453–467] accurately predicted purity and Kapp at pKapp values >4 and was independent of electrode characteristics. It is considered the method of choice.
Macroelectrode primary calibration should be carried out in solutions varying from 0.5 to 10 mmol l−1 combined with either Ca–EGTA or Mg–EDTA buffers; the [Ca2+] and [Mg2+] in other buffer ligands can be measured in a secondary calibration. 相似文献
The activity exhibited an absolute requirement for Mg2+ in the neutral pH range, while Ca2+ was found able to activate ATPase at more alkaline pH. Optimal activity was observed at pH 7.5, with a Mg/ATP ratio of 0.5.
ADP was found to inhibit ATP hydrolysis and to transform the Michaelian ATP concentration dependence with a Km of 0.5 mM into a sigmoid curve with increasing Km and decreasing V.
In contrast ADP activated an ATP-ADP exchange process and this shift from hydrolysis to exchange was stimulated by high Mg2+ and by orthophosphate.
All nucleoside triphosphates tested interfered with ATP hydrolysis, all could be hydrolyzed and could donate their terminal phosphate group to ADP. The relative efficiencies of nucleoside triphosphates in these three processes varied in parallel with minor discrepancies.
ATP hydrolysis was inhibited by N,N′-dicyclohexylcarbodiimide (DCCD) Dio 9, NaN3 and pyrophosphate, the first two being ineffective against ATP-ADP exchange, the third being stimulatory and the last inhibitory.
ATP hydrolysis and ATP-ADP exchange are tentatively attributed to the terminal enzyme of oxidative phosphorylation. 相似文献
At low ionic strength conditions the addition of Ni2+ induces a B → Z conformational transition in poly(dG-dC). As demonstrated by UV absorption and CD spectroscopy, the transition occurs at I = 0.001 M already when 3 × 10−5 – 7 × 10−5 M of Ni2+ are added to 8 × 10−5 M (in monomeric units) of poly(dG-dC), and at I = 0.01 M between 2.5 × 10−4 and 4.5 × 10−4 M of Ni2+. Using murexide as an indicator of the concentration of free Ni2+ ions, the amount of Ni2+ which is bound to the polynucleotide could be determined. At I = 0.001 M it was established that the B → Z transition begins when 1 Ni2+ is bound coordinatively per four base pairs, and the transition is complete when 1 Ni2+ is bound coordinatively per three base pairs. It is this coordinated Ni2+ which induces the B → Z transition. 相似文献
2. The amount of G-actin-bound calcium, as well as the sum of bivalent cation after replacement, not removable by short-time Dowex-50 treatment, accounts to about 1 mole per 50000 g of G-actin.
3. The rate of exchange is of the same order for bivalent cations studied, including calcium.
4. G-actin-bound Ca2+ is fully replaced, besides free Ca2+, by free Mn2+ and Cd2+. The replacement with Mg2+, Co2+, Ni2+ and Zn2+ is not complete, and there is practically no reaction with Ba2+ and Sr2+.
5. Assuming the affinity constant of Ca2+ as 1, the following affinity constants for other bivalent cations were obtained: Mn2+, 0.90; Cd2+, 1.07; Mg2+, 0.27; Zn2+, 0.22; Co2+, 0.18; Ni2+, 0.08.
6. The results obtained show that there exists a close correlation between the ionic radius of a particular bivalent cation, and its ability to replace bound Ca2+. 相似文献
2. In the presence of Mg2+ the nitreno-ATP is bound to both the and β subunits, mainly (63%) to the subunits.
3. After successive photolabelling of F1 with 8-azido-ATP (no Mg2+) and 8-azido-ADP (with Mg2+) 4 mol label is bound to F1, 2 mol to the and 2 mol to the β subunits.
4. When the order of photolabelling is reversed, much less 8-nitreno-ATP is bound to F1 previously labelled with 8-nitreno-ADP. It is concluded that binding to the -subunits hinders binding to the β subunits.
5. F1 that has been photolabelled with up to 4 mol label still contains 2 mol firmly bound adenine nucleotides per mol F1.
6. It is concluded that at least 6 sites for adenine nucleotides are present in isolated F1. 相似文献
1. 1. Tightly bound ATP and ADP, found on the isolated mitochondrial ATPase, exchange only slowly at pH 8, but the exchange is increased as the pH is reduced. At pH 5.5, more than 60% of the bound nucleotide exchanges within 2.5 min.
2. 2. Preincubation of the isolated ATPase with ADP leads to about 50% inhibition of ATP hydrolysis when the enzyme is subsequently assayed in the absence of free ADP. This effect, which is reversed by preincubation with ATP, is absent on the membrane-bound ATPase. This inhibition seems to involve the replacement of tightly bound ATP by ADP.
3. 3. Using these two findings, the binding specificity of the tight nucleotide binding sites was determined. iso-Guanosine, 2′-deoxyadenosine and formycin nucleotides displaced ATP from the tight binding sites, while all other nucleotides tested did not. The specificities of the tight sites of the isolated and membrane-bound ATPase were similar, and higher than that of the hydrolytic site.
4. 4. The nucleotide specificities of ‘coupled processes’ nucleoside triphosphate-driven reversal of electron transfer, nucleoside triphosphate-32Pi exchange and phosphorylation were higher than that of the hydrolytic site of the ATPase and similar to that of the tight nucleotide binding sites.
5. 5. The different nucleotide specificities of uncoupled ATP hydrolysis and coupled processes can be explained even if both processes involve a single common site on the ATPase molecule. This model requires that energy can be ‘coupled’ only when it is released/utilised in the nucleotide binding steps of the mechanism.
6. 6. Adenosine β,γ-imidotriphosphate (AMP-PNP) is not a simple reversible inhibitor of the ATPase, since incubation requires preincubation and is not reversed when the compound is diluted out, or by addition of ATP. This compound inhibits the isolated and membrane-bound ATPase equally well. Its guanosine analogue does not act in this way.
7. 7. In submitochondrial particles, ADP inhibited uncoupled hydrolysis of ATP much more effectively than coupled hydrolysis, the latter being measured both directly (from ATP hydrolysis in the absence of uncoupler) or indirectly, by monitoring ATP-driven reduction of NAD+ by succinate.
8. 8. The effects of ADP and AMP-PNP were interpreted as providing evidence for two of the intermediates in the proposed scheme for coupled triphosphate hydrolysis.
Abbreviations: ε-ATP, N1,N6-ethenoadenosine triphosphate; 8-BrATP, 8-bromoadenosine triphosphate; AMP-PNP, adenosine β,γ-imidotriphosphate; GMP-PNP, guanosine β,γ-imidotriphosphate; N1,O-ATP, adenosine-N1-oxide triphosphate; rro-ATP 2,2′[1-(9-adenyl)-1′-(triphosphoryl-oxymethyl)-dihydroxydiethyl ether; and similarly for the respective diphosphates; NTP, NDP, nucleoside tri-, diphosphate; ANS, 1-anilino-8-naphthalene sulphonate; FCCP, carbonylcyanide p-trifluoromethoxyphenylhydrazone; HEPES, N-2-hydroxyethylpiperazine-N′-2-ethane sulphonic acid; MES, 2-(N-morpholino)-ethane sulphonic acid; TES, tris(hydroxymethyl)methylamino ethane sulphonic acid 相似文献
(2) A light dependent increase in the Mg2+ content of the stroma was detected when chloroplasts were subjected to osmotic shock, amounting to 26 nmol/mg chlorophyll. Furthermore, a rapid and reversible light-dependent efflux of Mg2+ has been observed in intact chloroplasts when the divalent cation ionophore A 23 187 was added, indicating a light-dependent transfer of about 60 nmol of Mg2+ per mg chlorophyll from the thylakoid membranes to the stroma.
(3) CO2 fixation, but not phosphoglycerate reduction, could be completely inhibited when A 23 187 was added to intact chloroplasts in the absence of external Mg2+. If Mg2+ was then added to the medium, CO2 fixation was restored. Half of the maximal restoration was achieved with about 0.2 mM Mg2+, which is calculated to reflect a Mg2+ concentration in the stroma of 1.2 mM. The further addition of Ca2+ strongly inhibits CO2 fixation.
(4) The results suggest that illumination of intact chloroplasts causes an increase in the Mg2+ concentration of 1–3 mM in the stroma. Compared to the total Mg2+ content of chloroplasts, this increase is very low, but it appears to be high enough to have a possible function in the light regulation of CO2 fixation. 相似文献
The clostridial solubilized and membrane-bound ATPase showed different properties similar to the “allotopic” properties of mitochondrial and other bacterial ATPases. The membrane-bound ATPase in contrast to the soluble ATPase was sensitive to the ATPase inhibitor dicyclohexylcarbodiimide (DCCD). DCCD, at 10-4 M, led to 80% inhibition of the membrane-bound enzyme; oligomycin, ouabain, or NaN3 had no effect. The membrane-bound ATPase could not be stimulated by trypsin pretreatment.
Since none of the mono- or divalent cations had any truly stimulatory effect, and since a pH gradient (interior alkaline), which was sensitive to the ATPase inhibitor DCCD, was maintained during growth of C. pasteurianum, it was concluded that the function of the clostridial ATPase was the same as that of the rather similar mitochondrial enzyme, namely H+ translocation. A H+-translocating, ATP-consuming ATPase appears to be intrinsic equipment of all prokaryotic cells and as such to be phylogenetically very old; in the course of evolution the enzyme might have been developed to a H+-(re)translocating, ATP-forming ATPase as probably realized in aerobic bacteria, mitochondria and chloroplasts. 相似文献
2. EDTA does not appear to be bound by heart mitochondria. At neutral pH the chelator penetrates into the mitochondrial water volume to the same extent as sucrose and mannitol. At pH 8.1 where the removal of mitochondrial Mg2+ by EDTA is more effective, EDTA penetrates virtually the entire water volume. This penetration requires the presence of a source of energy, a transported cation such as Na+, and a permeant anion. It appears possible that the oscillations in ion uptake and swelling observed in the presence of EDTA at pH 8.1 may be related to the presence of the chelator in the interior compartment under these conditions. 相似文献