共查询到20条相似文献,搜索用时 15 毫秒
1.
Elena Toll Pierre Crespi Hubert Greppin F. J. Castillo 《Trees - Structure and Function》1998,13(1):13-18
One- and 2-year-old Pinus sylvestris saplings were exposed to chronic doses of ozone (O3) and sulphur dioxide (SO2) in short-term (3 months) and long-term (18 months) experiments. Microsomal and plasma membrane fractions were purified by
phase partitioning from current-year needles. The following membrane enzyme activities were determined in the microsomal and/or
purified plasma membrane fractions: K+, Mg2+-ATPase (EC 3.6.1.3), NADH ferricyanide oxidoreductase (EC 1.6.99.3), NADH-duroquinone reductase (EC 1.6.5.1), NADH oxidase
type I (EC 1.6.99.2), NADH oxidase type II or peroxidase-like enzyme (EC 1.11.1.7) and pyrophosphatase (EC 3.6.1.1). NADH
oxidase type I was slightly stimulated in the microsomal fraction after a short-term exposure to O3 whereas NADH-dependent duroquinone reductase was not affected by this pollutant. However, in the long term experiment, NADH
oxidase type II measured in the plasma membrane fraction was more than 2-fold stimulated in the SO2 treated pines and more than 4-fold when O3 was added to SO2. However, pyrophosphatase was decreased by 50% in trees treated with SO2+O3 and remained unchanged in the SO2 treatment, indicating that this enzyme is probably sensitive to oxidation. K+, Mg2+-ATPase showed a trend towards an enhancement of activity when exposed to chronic concentrations of air pollutants, this enhancement
was more important in the long-term experiment after the combined effect of SO2 and O3. However, the K+-stimulated component was inhibited by the combination of both pollutants. Finally, NADH ferricyanide reductase was significantly
enhanced after O3 and SO2+O3 exposures appearing as the most sensitive oxidoreductase to these air pollutants. The stimulation of ATPase and membrane
oxidoreductases could facilitate the adaptation and defense of trees by maintaining an adequate redox potential in the plasma
membrane region and perhaps stimulating the reduction of extracellular electron acceptors generated by the exposure to air
pollutants.
Received: 15 September 1997 / Accepted: 4 May 1998 相似文献
2.
Effects of T3- and T4-induced thyrotoxicosis on temperature-dependent Arrhenius kinetics of succinate oxidase and Mg2+- and Mg2+ + 2,4-dinitrophenol-dependent ATPase activities in rat heart mitochondria were examined, For succinate oxidase system, treatment
with T3 and T4 caused increase in the energy of activation in high temperature range in a dose-dependent manner. For low temperature range,
increase in energy of activation was apparent only with higher doses of the hormones; with low doses a small but reproducible
decrease was evinced. The phase transition temperature decreased significantly under these conditions. For the Mg2+- and Mg2++2,4-dintro-phenol-dependent-ATPase activities, the activation energy values in high temperature range decreased in general.
Activation energy values in low temperature range recorded a generalized increase in the Mg2+-ATPase enzyme system while the value did not change significantly for the Mg2+ + 2,4-dinitrophenol-ATPase; phase transition temperature registered a small but reproducible decrease under these conditions.
The results are suggestive of increased membrane fluidization possibly through increased proportion of unsaturated fatty acids.
The differential effects seen for succinate oxidase and ATPase systems are consistent with different lipid protein domains
of these enzyme systems. 相似文献
3.
The membrane sector (F0) of H+-ATPase was prepared by trypsin and urea treatment of F1-F0 and reconstituted with purified F1. The oligomycin sensitivity of the reconstituted F1-F0 complex obtained by treating F1 or F0 with Mg2+ before binding is much higher than that obtained without Mg2+ treatment. The greater change in the intrinsic fluorescence of the reconstituted F1-F0 complex obtained by Mg2+ treatment suggests that conformational changes may occur during the reconstitution. We deduce that Mg2+ binds to membrane lipids, thus decreasing membrane fluidity and changing the physical state of the lipids to provide a suitable microenvironment for conformational changes in F0. The data also suggest that the conformational change in the F0 portion of the F1-F0 complex can be transmitted to the F1 portion, the conformation of which is in turn altered, resulting in the formation of an F1-F0 complex with high oligomycin sensitivity. On the other hand, Mg2+ may act on F1 directly to induce a suitable conformational change which is then trnsmitted to F0, resulting in the formation of an H+-ATPase with greater sensitivity to oligomycin.Abbreviations STED
0.25 M sucrose, 10 mM Tris-SO4, 0.2 mM EDTA, and 1 mM dithiothreitol, pH 8.0
- NADH
nicotinamide adenine dinucleotide, reduced form
- olig.
oligomycin
- OSCP
oligomycin sensitivity conferring protein
- F6
coupling factor 6
- F1
coupling factor one (or F1-ATPase)
- F1
+Mg
2+ and F1
–Mg
2+
the F1 treated and untreated with 1 mM Mg2+ respectively
- F0
the membrane sector proteins of the H+-ATPase
- TUF0
trypsin-urea – F0
- EUF0
EDTA-urea – F0
- F0
+Mg
2+ and F0
–Mg
2+
the F0 treated and untreated with 1 mM Mg2+ respectively
- (F1 · F0)+Mg
2+ and (F1 · F0)–Mg
2+
the reconstituted F1 · F0 complex containing Mg2+-treated F1 and F0 and untreated F1 and F0 respectively
- F1 · F0
+Mg
2+ and F1 · F0
–Mg
2+
the reconstituted H+-ATPase complex derived from the binding of purified F1 to the F0 treated and untreated with Mg2+ respectively
- F1
+Mg
2+ · F0 and F1
–Mg
2+ · F0
the reconstituted H+-ATPase derived from the binding of F0 to the purified F1 treated and untreated with Mg2+ respectively 相似文献
4.
Summary Conformational states in sarcoplasmic reticulum Ca2+-ATPase have been examined by tryptic and chymotryptic cleavage. High affinity Ca2+ binding (E1 state) exposes a peptide bond in the A fragment of the polypeptide chain to trypsin. Absence of Ca2+ (E2 state) exposes bonds in the B fragment, which are protected by binding of Mg2+ or ATP. After phosphorylation from ATP the tryptic cleavage pattern depends on the predominant phosphoenzyme species present. ADP-sensitive E1P and ADP-insensitive E2P have cleavage patterns identical to those of unphosphorylated E1 and E2, respectively, indicating that two major conformational states are involved in Ca2+ translocation. The transition from E1P to E2P is inhibited by secondary tryptic splits in the A fragment, suggesting that parts of this fragment are of particular importance for the energy transduction process.The tryptic cleavage patterns of phosphorylated forms of detergent solubilized monomeric Ca2+-ATPase were similar to those of the membrane-bound enzyme, indicating that Ca2+ translocation depends mainly on structural changes within a single peptide chain. On the other hand, the protection of the second cleavage site as observed after vanadate binding to membranous Ca2+-ATPase could not be achieved in the soluble monomeric enzyme. Shielding of this peptide bond may therefore be due to protein-protein interactions in the semicrystalline state of the vanadate-bound Ca2+-ATPase in membranous form. 相似文献
5.
Rita Sikdar Uma Ganguly Pratima Pal Barsanjit Mazumder Parimal C. Sen 《Molecular and cellular biochemistry》1991,103(2):121-130
The goat spermatozoa membranes isolated after treatment with octa (ethylene glycol) mono n-dodecyl ether (C12E8) followed by discontinuous sucrose density gradient centrifugation have been found to contain an ATPase that is stimulated by externally added Ca2+ only. The membrane fraction has also found to contain Mg2+-dependent Ca2+-ATPase activity, however the former activity is about 2 fold higher than the latter. The molecular weight of the enzyme is found to be about 97,000 on SDS-polyacrylamide gel. The optimum concentration of Ca2+ required for maximum activity is 3 mM for both Mg2+-dependent and Mg2+-independent Ca2+-ATPase. Histidine and imidazole buffers are found to be the most suitable for dependent and independent enzyme activities respectively. ATP with an optimum concentration of 4 mM is observed to be the best substrate than any other nucleotides. The inhibitors like trifluoperazine and vanadate and group specific probes e.g. DTNB and TNBS inhibit these two enzymes but at different rates. Ca2+-uptake study shows that the uptake in the presence of Ca2+ and ATP is higher than in the presence of Mg2+, Ca2+ and ATP. The findings lead us to believe that the Mg2+-independent Ca2+-ATPase has some role in Ca2+ transport like Mg2+-dependent enzyme.Abbreviations Tris
Tris (hydroxymethyl) amino ethane
- Hepes-N
2-hydroxy ethyl piperizine-N1-2-ethane sulfonic acid
- Pipes-Piperizine-N
N1-bis(2-ethane sulfonic acid)
- EGTA
Ethylene Glycol-bis (-amino ethyl ether)
- N, N, N1, N1
Tetraacetic Acid, sodium salt
- TFP
Trifluoperazine
- DTNB
5,51 Dithiobis (2 nitrobenzoic acid)
- TNBS
2, 4, 6-Trinitrobenzene Sulfonate
- C12E8
Octa (ethylene glycol) mono n-dodecyl ether
- PMSF
Phenylmethyl Sulfonyl Fluoride
- PAGE
Polyacrylamide Gel Electrophoresis
- PME
-Mercapto Ethanol 相似文献
6.
Eguchi H Morii M Takahashi Y Sakai H Nakano M Ochiai H Shirahata A Hara Y Kawamura M Takeda K 《The Journal of membrane biology》2008,221(3):133-140
Leucines were mutated within the sequence L311ILGYTWLE319 of the extracellular loop flanking the third (M3) and fourth (M4) transmembrane segments (M3/M4 loop) of the Torpedo Na+,K+-ATPase α-subunit. Replacement of Leu311 with Glu resulted in a considerable loss of Na+,K+-ATPase activity. Replacement of Leu313 with Glu shifted the equilibrium of E1P and E2P toward E1P and reduced the rate of the E1P to E2P transition. The reduction of the transition rate and stronger inhibition of Na+,K+-ATPase activity by Na+ at higher concentrations together suggest that there is interference of Na+ release on the extracellular side in the Leu313 mutant. Thus, Leu313 could be in the pathway of Na+ exit. Replacement of Leu318 with Glu yielded an enzyme with significantly reduced apparent affinity for both vanadate and K+, with an equilibrium shifted toward E2P and no alteration in the transition rate. The reduced vanadate affinity is due to the lower rate of production of vanadate-reactive
[K+
2]E2 caused by inhibition of dephosphorylation through reduction of the K+ affinity of E2P. Thus, Leu318 may be a critical position in guiding external K+ to its binding site. 相似文献
7.
Plasma membranes were isolated after binding liver and hepatoma cells to polylysine-coated polyacrylamide beads, and the effect of concanavalin A on the membrane-bound Mg2+-ATPase and the Mg2+-ATPase solubilized by octaethylene glycol monododecyl ether (C12E8) was studied. In the experiment of membranebound Mg2+-ATPase, plasma membranes were pretreated with Concanavalin A and the activity was assayed. Concanavalin A stimulated the activity of both liver and hepatoma enzymes assayed above 20°C. Concanavalin A abolished the negative temperature dependency characteristic of liver plasma membrane Mg2+-ATPase. On the other hand, Concanavalin A prevented the rapid inactivation due to storage at ?20°C, which was characteristic of hepatoma plasma membrane Mg2+-ATPase. With solubilized Mg2+-ATPase from liver plasma membranes, the negative temperature dependency was not observed. Concanavalin A, which was added to the assay medium, stimulated the activity of the enzyme solubilized in C12E8 at a high ionic strength. However, Concanavalin A failed to show any effect on the enzyme solubilized in C12E8 at a low ionic strength. With solubilized Mg2+-ATPase from hepatoma plasma membranes, Concanavalin A could not prevent the inactivation of the enzyme during incubation at ?20°C. 相似文献
8.
Peter Leth Jorgensen Jesper R. Jorgensen Per Amstrup Pedersen 《Journal of bioenergetics and biomembranes》2001,33(5):367-377
In the P-domain, the 369-DKTGTLT and the 709-GDGVNDSPALKK segment are highly conserved during evolution of P-type E1–E2-ATPase pumps irrespective of their cation specificities. The focus of this article is on evaluation of the role of the amino acid residues in the P domain of the subunit of Na,K-ATPase for the E1P[3Na] E2P[2Na] conversion, the K+-activated dephosphorylation, and the transmission of these changes to and from the cation binding sites. Mutations of residues in the TGDGVND loop show that Asp710 is essential, and Asn713 is important, for Mg2+ binding and formation of the high-energy MgE1P[3Na] intermediate. In contrast Asp710 and Asp713 do not contribute to Mg2+ binding in the E2P–ouabain complex. Transition to E2P thus involves a shift of Mg2+ coordination away from Asp710 and Asn713 and the two residues become more important for K+-activated hydrolysis of the acyl phosphate bond at Asp369. Transmission of structural changes between the P-domain and cation sites in the membrane domain is evaluated in light of the protein structure, and the information from proteolytic or metal-catalyzed cleavage and mutagenesis studies. 相似文献
9.
Summary The plasma membrane (Mg2+)-dependent adenosine triphosphatase ((Mg2+)-ATPase) from human erythrocytes has been tested for its ability to transport ions. Using a preparation of inside-out vesicles loaded with the pH-sensitive fluorescence probe 1-hydroxypyrene-3,6,8-trisulfonic acid (HPTS), we have demonstrated the absence of proton movement during (Mg2+)-ATPase activity. From the rate of ATP hydrolysis and the passive proton permeability of these vesicles, an upper limit of 0.03 H+ transported per ATP hydrolyzed was calculated. To verify that proton pumping could be detected in this system, the intravesicular pH was monitored during (Ca2+)-dependent adenosine triphosphatase ((Ca2+)-ATPase) activity. Proton efflux associated with (Ca2+)-ATPase activity was observed (in agreement with a recent report of proton pumping by a reconstituted erythrocyte (Ca2+)-ATPase (Niggli, V., Sigel, E., Carafoli, E. (1982)J. Biol. Chem.
257:2350–2356)) and was shown to be stimulated by calmodulin. The ability of the (Mg2+)-ATPase to pump28Mg2+,35SO
4
2–
and86Rb+ was also tested, with the results leading to the conclusion that the human erythrocyte enzyme does not function as an ion transport system. 相似文献
10.
The number of accessible SH groups was determined in membrane vesicles prepared from Enterococcus hirae grown under anaerobic conditions at alkaline pH (pH 8.0). Addition of ATP or nicotinamide adenine dinucleotides (NAD++NADH) to the vesicles caused a ∼4-fold or ∼1.9-fold increase in the number of SH-groups, respectively. This was inhibited
by treatment with N-ethylmaleimide. The increase was significant when ATP and NAD++NADH both were added. The change was lacking in the presence of the F0F1-ATPase inhibitors N,N′-diclohexylcarbodiimide or sodium azide. This was also absent in atp mutant with defect in the F0F1-ATPase and, in addition, it was less in potassium ion–free medium. These results are correlated with data about K+-dependent F0F1-ATPase activity, suggesting a relationship between the F0F1-ATPase and K+ uptake Trk-like system. The latter may be regulated by NAD or NADH mediating conformational changes. 相似文献
11.
The regulatory effects of malate on chloroplast Mg2+-ATPase were investigated and the mechanism was discussed. Malate stimulated methanol-activated membrane-bound and isolated CF1 Mg2+-ATPase activity. The subunit of CF1 may be involved in malate regulation of the enzyme function. Modification of subunit at one site of the peptide by NEM may affect malate stimulation of ATPase while at another site may have no effect. The effect of malate on the Mg2+-ATPase was also controlled by the Mg2+/ATP ratio in the reaction medium. The enhancing effect of malate on Mg2+-ATPase activity depended on the presence of high concentrations of Mg2+ in the reaction mixture. Kinetic study showed that malate raised the Vmax of catalysis without affecting the Km for Mg2+ ATP. The experiments imply that the stimulation of Mg2+-ATPase by malate is probably correlated with the Pi binding site on the enzyme. The regulation of ATPase activity by malate in chloroplasts may be relevant to its function in vivo.Abbreviations CF1
chloroplast coupling factor 1
- CF1 (-) and CF1 (-)
CF1 deficient in the and subunit
- MF1
mitochondria coupling factor 1
- NEM
N-ethylmaleimide
- PMS
phenazine methosulfate
- OG
n-octyl--d-glucopyranoside 相似文献
12.
Rivka Goldshleger Guy Patchornik Meirav Bar Shimon Daniel M. Tal Robert L. Post Steven J. D. Karlish 《Journal of bioenergetics and biomembranes》2001,33(5):387-399
This chapter describes contributions of transition metal-catalyzed oxidative cleavage of Na+,K+-ATPase to our understanding of structure–function relations. In the presence of ascorbate/H2O2, specific cleavages are catalyzed by the bound metal and because more than one peptide bond close to the metal can be cleaved, this technique reveals proximity of the different cleavage positions within the native structure. Specific cleavages are catalyzed by Fe2+ bound at the cytoplasmic surface or by complexes of ATP–Fe2+, which directs the Fe2+ to the normal ATP–Mg2+ site. Fe2+- and ATP–Fe2+-catalyzed cleavages reveal large conformation-dependent changes in interactions between cytoplasmic domains, involving conserved cytoplasmic sequences, and a change of ligation of Mg2+ ions between E1P and E2P, which may be crucial in facilitating hydrolysis of E2P. The pattern of domain interactions in E1 and E2 conformations, and role of Mg2+ ions, may be common to all P-type pumps. Specific cleavages can also be catalyzed by Cu2+ ions, bound at the extracellular surfaces, or a hydrophobic Cu2+-diphenyl phenanthroline (DPP) complex, which directs the Cu2+ to the membrane–water interface. Cu2+- or Cu2+-DPP-catalyzed cleavages are providing information on / subunit interactions and spatial organization of transmembrane segments. Transition metal-catalyzed cleavage could be widely used to investigate other P-type pumps and membrane proteins and, especially, ATP binding proteins. 相似文献
13.
K.Damodar Reddy A Chaudhuri K Thangavelu 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》1997,116(4):459-466
Tissue-specific age-dependent changes were observed in Na+K+-, Ca2+-, and Mg2+-ATPase activities in tropical tasar silkworm, Antheraea mylitta Drury. Maximum enzyme activity was recorded in all the tissues on day 12 (before spinning) in control group of animals. In testis, Na+K+-, Ca2+-, and Mg2+-ATPase activities gradually increased from day 2 to day 12 during fifth larval age and level was maintained up to adult eclosion while, in ovary, a marked decline was noted up to day of adult emergence. Further, a significant and sharp rise was found in ATPase activity in silk gland tissue up to day 12 and afterwards a drastic fall was noted on day 15 (end of spinning) during fifth larval age.Administration of T4 to fifth stage larvae (1 hr old) at doses 0.5–2.0 μg/g significantly elevated the Na+K+-, Ca2+-, and Mg2+-ATPase activities in larval and pupal gonads in a dose-dependent fashion. But, in moths, the enhancement was very much confined to Na+K+- and Ca2+-ATPase in testes and only Ca2+-ATPase in ovaries. Again, in silk glands thyroxine (0.5–2.0 μg/g) caused a significant rise in the all ion-dependent ATPase activities only during the fifth larval stage. Interestingly, higher doses of T4 (4.0 μg/g) caused a significant reduction in Na+K+-, Ca2+- and Mg2+-ATPase in all the tissues almost all the days studied so far. However, lower doses of T4 (0.1 and 0.25 μg/g) remained ineffective in altering the different ion-specific ATPase activities. This study suggests, that mammalian thyroxine has a metabolic influence showing biphasic nature of action in tasar silkworm ATPase system. 相似文献
14.
F.M.A.H. Schuurmans Stekhoven H.G.P. Swarts J.J.H.H.M. de Pont S.L. Bonting 《生物化学与生物物理学报:生物膜》1981,649(3):533-540
ATP and adenylylimidodiphosphate (AdoPP[NH]P) bind to (Na+ + K+)-ATPase in the absence of Mg2+ (EDTA present) with a homogeneous but 15-fold different affinity, the Kd values being 0.13 μM and 1.9 μM, respectively. The binding capacities of the two nucleotides are nearly equal and amount to 3.9 and 4 nmol/mg protein or 1.7 and 1.8 mol/mol (Na+ + K+)-ATPase, respectively. The Kd value for ATP is equal to the Km for phosphorylation by ATP (0.05–0.25 μM) and the binding capacity is equivalent to the phosphorylation capacity of 1.8 mol/mol (Na+ + K+)-ATPase. Hence, the enzyme contains two high-affinity nucleotide binding and phosphorylating sites per molecule, or one per α-subunit. Additional low-affinity nucleotide binding sites are elicited in the presence of Mg2+, as shown by binding studies with the non-phosphorylating (AdoPP[NH]P). The Kd and binding capacity for AdoPP[NH]P at these sites is dependent on the Mg2+ concentration. The Kd increases from 0.06 mM at 0.5 mM Mg2+ to a maximum of 0.26 mM at 2 mM Mg2+ and the binding capacity from 1.5 nmol/mg protein at 0.5 mM Mg2+ to 3.3 nmol/mg protein at 4 mM Mg2+. Extrapolation of a double reciprocal plot of binding capacity vs. total Mg2+ concentration yields a maximal binding capacity at infinite Mg2+ concentration of 3.8 nmol/mg protein or 1.7 mol/mol (Na+ + K+)-ATPase. The Kd for Mg2+ at the sites, where it exerts this effect, is 0.8 mM. The Kd for the high-affinity sites increases from 1.5–1.9 μM in the absence of Mg2+ to a maximum of 4.2 μM at 2 mM Mg2+ concentration. The binding capacity of these sites (1.8 mol/mol enzyme) is independent of the Mg2+ concentration. Hence, Mg2+ induces two low-affinity non-phosphorylating nucleotide binding sites per molecule (Na+ + K+)-ATPase in addition to the two high-affinity, phosphorylating nucleotide binding sites. 相似文献
15.
S. N. Nagendra K. Mamatha Rao M. N. Subhash K. Taranath Shetty 《Neurochemical research》1994,19(12):1509-1513
The chronic administration of disulfiram (DS) to rats resulted in significant decrease of synaptosomal Ca2+, Mg2+-ATPase activity. In vitro studies indicated that DS (ID50=20 M) produced a dose-dependent inhibition of Ca2+, Mg2+-ATPase. However, diethyldithio-carbamate, a metabolite of DS, failed to modify Ca2+, Mg2+-ATPase activity, implying that the decrease in ATPase activity in DS administered rats was due to the effect of parent compound. The DS-mediated inhibition (48%) of ATPase activity was comparable with a similar degree of inhibition (49%) achieved by treating the synaptosomal membranes with N-ethylmaleimide (ID50=20 M) in vitro. Furthermore, the inhibition by DS was neither altered by washing the membranes with EGTA nor reversed by treatment with sulfhydryl reagents such as GSH or dithiothreitol. About 74% and 68% decrease of synaptosomal Ca2+, Mg2+-ATPase specific activity was observed when treated with DS (30 M) and EGTA (100 M) respectively. The remaining 25–30% of total activity is suggested to be of Mg2+-dependent ATPase activity. This indicates that both these drugs may act on a common target, calmodulin component that represents 70–75% of total Ca2+, Mg2+-ATPase activity. Therefore, DS-mediated modulation of synaptosomal Ca2+, Mg2+-ATPase activity could affect its function of maintaining intracellular Ca2+ concentration. This could contribute to the deleterious effects on CNS. 相似文献
16.
B.S. Jena B.K. Patnaik 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》1995,111(4):545-552
Hepatic Na+-K+-ATPase and Mg2+-ATPase activities of male green lizards declined during the maturation phase (juvenile to 1-year-old) and stabilized thereafter. On the other hand, the Ca2+-ATPase activity of the liver declined during the later half of the life span (1-year-old to 2–4-year-old). Starvation stress induced a decline in hepatic Na+-K+-ATPase and Mg2+-ATPase activities of juvenile lizards and caused an increase in 1-year-old and 2–4-year-old counterparts. The Ca2+-ATPase activity declined only in starved 1-year-old lizards. Following cold stress, the hepatic Na+-K+-ATPase activity of juvenile lizards showed a higher degree of decline than 2–4-year-old counterparts. The Mg2+-ATPase activity declined in cold-stressed juvenile lizards, but the parameter was elevated in similarly treated 1-year-old lizards. On the other hand, the increase in Ca2+-ATPase activity in response to cold stress was confined only to 2–4-year-old lizards. 相似文献
17.
H.K. Kimelberg 《生物化学与生物物理学报:生物膜》1975,413(1):143-156
The (Na++K+)-activated, Mg2+-dependent ATPase from rabbit kidney outer medulla was prepared in a partially inactivated, soluble from depleted of endogenous phospholipids, using deoxycholate. This preparation was reactivated 10 to 50-fold by sonicated liposomes of phosphatidylserine, but not by non-sonicated phosphatidylserine liposomes or sonicated phosphatidylcholine liposomes. The reconstituted enzyme resembled native membrane preparations of (Na++K+)-ATPase in its pH optimum being around 7.0 showing optimal activity at Mg2+: ATP mol ratios of approximately 1 and a Km value for ATP of 0.4 mM.Arrhenius plots of this reactivated activity at a constant pH of 7.0 and an Mg2+: ATP mol ratio of 1:1 showed a discontinuity (sharp change of slope) at 17 °C, With activation energy (Ea) values of 13–15 kcal/mol above this temperature and 30–35 kcal below it. A further discontinuity was also found at 8.0 °C and the Ea below this was very high (> 100 kcal/mol).Incresed Mg2+ concentrations at Mg2+: ATP ratios in excess of 1:1 inhibited the (Na++K+)-ATPase activity and also abolished the discontinuities in the Arrhenius plots.The addition of cholesterol to phosphatidylserine at a 1:1 mol ratio partially inhibited (Na++K+)-ATPase reactivation. Arrhenius plots under these conditions showed a single discontinuity at 20°C and Ea values of 22 and 68kcal/mol above and below this temperature respectively. The ouabain-insensitive Mg2+-ATPase normally showed a linear Arrhenius plot with an Ea of 8 kcal/mol. The cholesterol-phosphatidylserine mixed liposomes stimulated the Mg2+-ATPase activity, which now also showed a discontinuity at 20 °C with, however, an increased value of 14 kcal/mol above this temperature and 6 kcal/mol below. Kinetic studies showed that cholesterol had no significant effect on the Km for ATP.Since both of cholesterol and Mg2+ are know to alter the effects of temperature on the fluidity of phospholipids the above result are discussed in this context. 相似文献
18.
Pontes ZL Oliveira LS Franzon R Wajner M Wannmacher CM Wyse AT 《Neurochemical research》2001,26(12):1321-1326
Na+,K+-ATPase and Mg2+-ATPase activities were determined in the synaptic plasma membranes from hippocampus of rats subjected to chronic and acute proline administration. Na+,K+-ATPase activity was significantly reduced in chronic and acute treatment by 33% and 40%, respectively. Mg2+-ATPase activity was not altered by any treatment. In another set of experiments, synaptic plasma membranes were prepared from hippocampus and incubated with proline or glutamate at final concentrations ranging from 0.2 to 2.0 mM. Na+,K+-ATPase, but not Mg2+-ATPase was inhibited (30%) by the two amino acids. In addition, competition between proline and glutamate for the enzyme activity was observed, suggesting a common binding site for these amino acids. Considering that Na+,K+-ATPase activity is critical for normal brain function, the results of the present study showing a marked inhibition of this enzyme by proline may be associated with the neurological dysfunction found in patients affected by type II hyperprolinemia. 相似文献
19.
- 1. (1) VO3− combines with high affinity to the Ca2+-ATPase and fully inhibits Ca2+-ATPase and Ca2+-phosphatase activities. Inhibition is associated with a parallel decrease in the steady-state level of the Ca2+-dependent phosphoenzyme.
- 2. (2) VO3− blocks hydrolysis of ATP at the catalytic site. The sites for VO3− also exhibit negative interactions in affinity with the regulatory sites for ATP of the Ca2+-ATPase.
- 3. (3) The sites for VO3− show positive interactions in affinity with sites for Mg2+ and K+. This accounts for the dependence on Mg2+ and K+ of the inhibition by VO3−. Although, with less effectiveness, Na+ substitutes for K+ whereas Li+ does not. The apparent affinities for Mg2+ and K+ for inhibition by VO3− seem to be less than those for activation of the Ca2+-ATPase.
- 4. (4) Inhibition by VO3− is independent of Ca2+ at concentrations up to 50 μM. Higher concentrations of Ca2+ lead to a progressive release of the inhibitory effect of VO3−.
Keywords: Ca2+-ATPase; Vanadate inhibition; K+; Li+; (Red cell membrane) 相似文献
20.
Some kinetic parameters of the human red cell Ca2+-ATPase were studied on calmodulin-free membrane fragments following preincubation at 37°C. After 30 min treatment with EGTA(1 mm) plus dithioerythritol (1 mm), a V max of about 0.4 μmol Pi/mg × hr and a K s of 0.3 μm Ca2+ were found. When Mg2+ (10 mm) or Ca2+(10 μm) were also added during preincubation, V maxbut not Kwas altered. Ca2+ was more effective than Mg2+, thus increasing V max to about 1.3 μmol Pi/mg × hr. The presence of both Ca2+ and Mg2+ during pretreatment decreasedKto 0.15 μm, while having no apparent effect on V max. Conversely, addition of ATP (2 mm) with either Ca2+ or Ca2+ plus Mg2+increased Vmax without affecting K. Preincubation with Ca2+ for periods longer than 30 min further increased Vmaxand reduced Kto levels as low as found with calmodulin treatment. The Ca2+ activation was not prevented by adding proteinase inhibitors (iodoacetamide, 10 mm; leupeptin, 200 μm; pepstatinA, 100 μm; phenylmethanesulfonyl fluoride, 100 μm). The electrophoretic pattern of membranes preincubated with or without Mg2+, Ca2+ or Ca2+ plus Mg2+ did not differ significantly from each other. Moreover, immunodetection of Ca2+-ATPase by means of polyclonal antibodiesrevealed no mobility change after the various treatments. The above stimulation was not altered by neomycin (200 μm), washing with EGTA (5 mm) or by both incubating and washing with delipidized serum albumin (1 mg/ml), or omitting dithioerythritol from the preincubation medium. On the other hand, the activation elicited by Ca2+ plus ATP in the presence of Mg2+ was reduced 25–30% by acridine orange (100 μm), compound 48/80 (100 μm) or leupeptin (200 μm) but not by dithio-bis-nitrobenzoic acid (1 mm). The fluorescence depolarization of 1,6-diphenyl-and l-(4-trimethylammonium phenyl)-6-phenyl 1,3,5-hexatriene incorporated into membrane fragments was not affected after preincubating under the different conditions. The results show that proteolysis, fatty acid production, an increased phospholipid metabolism or alteration of membrane fluidity are not involved in the Ca2+ effect. Ca2+ preincubation may stimulate the Ca2+-ATPase activity by stabilizing or promoting the E1 conformation. 相似文献