首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 537 毫秒
1.
Isolated porcine platelet α granules display a Mg2+-stimulated ATPase activity. The enzyme is membrane bound and several criteria suggest that it is intrinsic to the α granules, rather than arising from contamination with other structures. Characterization of the ATPase revealed an apparent Km for ATP of 198 μm. Other nucleotides are also hydrolyzed by the enzyme, though at a slower rate. The enzyme has an absolute requirement for divalent cations, and both Mg2+ (apparent Km 0.93 mm) and Ca2+ (apparent Km 0.95 mm) can activate it. Maximal hydrolysis rates are higher with Mg2+ than with Ca2+. Micromolar Ca2+ in the presence of maximally stimulating Mg2+ concentrations produces a small additional enhancement of activity. The Mg2+ ATPase has a broad activity maximum between pH 6.5 and 8.5, and an activation energy of 11.8 Kcal/mol. Several independent observations suggest that the ATPase could be involved in H+ translocation across the granule membrane: (a) the activity is stimulated upon disrupting membrane continuity by either hypotonic lysis or addition of nondenaturing detergents; (b) proton ionophores enhance the activity in intact but not in disrupted α granules; (c) permeating anions stimulate the ATPase more than slowly permeant or impermeant ones; (d) addition of NH3 (as either NH4Cl or (NH4)2SO4) activates enzyme activity; (e) silicotungstate and disulfonic stilbene derivatives, which are inhibitors of other H+-transporting ATPases, also inhibit the α-granule enzyme. These findings are compared with the reported properties of H+ pumps of other storage and secretory organelles.  相似文献   

2.
Abstract: With a partially purified, membrane-bound (Ca + Mg)-activated ATPase preparation from rat brain, the K0.5 for activation by Ca2+ was 0.8 p μm in the presence of 3 mm -ATP, 6 mm -MgCl2, 100 mM-KCI, and a calcium EGTA buffer system. Optimal ATPase activity under these circumstances was with 6-100 μm -Ca2+, but marked inhibition occurred at higher concentrations. Free Mg2+ increased ATPase activity, with an estimated K0.5, in the presence of 100 μm -CaCl2, of 2.5 mm ; raising the MgCl2 concentration diminished the inhibition due to millimolar concentrations of CaCl2, but antagonized activation by submicromolar concentrations of Ca2+. Dimethylsulfoxide (10%, v/v) had no effect on the K0.5 for activation by Ca2+, but decreased activation by free Mg2+ and increased the inhibition by millimolar CaCl2. The monovalent cations K+, Na+, and TI+ stimulated ATPase activity; for K+ the K0.5 was 8 mm , which was increased to 15 mm in the presence of dimethylsulfoxide. KCI did not affect the apparent affinity for Ca2+ as either activator or inhibitor. The preparation can be phosphorylated at 0°C by [γ-32P]-ATP; on subsequent addition of a large excess of unlabeled ATP the calcium dependent level of phosphorylation declined, with a first-order rate constant of 0.12 s?1. Adding 10 mm -KCI with the unlabeled ATP increased the rate constant to 0.20 s?1, whereas adding 10 mm -NaCl did not affect it measurably. On the other hand, adding dimethyl-sulfoxide slowed the rate of loss, the constant decreasing to 0.06 s?1. Orthovanadate was a potent inhibitor of this enzyme, and inhibition with 1 μm -vanadate was increased by both KCI and dimethylsulfoxide. Properties of the enzyme are thus reminiscent of the plasma membrane (Na + K)-ATPase and the sarcoplasmic reticulum (Ca + Mg)-ATPase, most notably in the K+ stimulation of both dephosphorylation and inhibition by vanadate.  相似文献   

3.
Europium luminescence from europium bound to sarcoplasmic reticulum (Ca2+ Mg2+)-ATPase indicates that there are two high affinity calcium binding sites. Furthermore, the two calcium ions at the binding sites are highly coordinated by the protein as the number of H2O molecules surrounding the Ca2+ ions are 3 and 0.5. In the presence of ATP, calcium ions are occluded even further down to 2 and zero H2O molecules, respectively. The Ca2+ - Ca2+ intersite distance is estimated to be 8–9 Å and the average distance from the Ca2+ sites to CrATP is about 18 Å.Digestion of the (Ca2+ + Mg2+)-ATPase at the T2 site (Arg 198) causes uncoupling of Ca2+-transport from ATPase activity while calcium occlusion due to E1-P formation remains unchanged. Further tryptic digestion beyond T2 and in the presence of ATP diminishes Ca2+ occlusion to zero while 50% of the ATPase hydrolytic activity remains. Tryptic digestion beyond T2 and in the absence of ATP diminishes ATPase hydrolytic activity to 50% of normal while Ca2+ occlusion remains intact. These data are consistent with a mechanism in which the functional enzyme must be in the dimeric form for occlusion and calcium uptake to occur, but each monomer can hydrolyze ATP.  相似文献   

4.
《Life sciences》1997,60(20):PL289-PL294
Therapeutic concentrations of praziquantel produce a rapid and intense contraction of the human flatworm Schistosoma mansoni. As an action on ATPases responsible for calcium homeostasis arises as a possible explanation for the molecular mechanism of this effect, we tested here the effect of praziquantel on different preparations from male adult worms that were previously characterized for their content in (Na++K+)-ATPase and (Ca2+-Mg2+)ATPase activities from different origins. Concentrations as high as 100 μM praziquantel did not inhibit (Na++K+)-ATPase from tegument and carcass nor (Ca2+-Mg 2+)ATPase from heterogeneous (P1) and microsomal (P4) fractions. As 100 μM praziquantel was also without effect on calcium permeability of microsomal vesicles actively loaded with 45Ca2+, the present results discard three hypotheses recently raised for the mechanism of praziquantel-induced contraction of S. mansoni.  相似文献   

5.
Horse muscle acylphosphatase (EC 3.6.1.7) was found to hydrolyze the labeled phosphorylated intermediate of (Ca2+ + Mg2+)-ATPase from rabbit muscle. In addition, the phosphorylated peptides obtained by pepsin digestion of the labeled phosphorylated microsomes were completely hydrolyzed by acylphosphatase. These findings suggest a possible regulatory role of this enzyme in vivo on the calcium transport process by sarcoplasmic reticulum.  相似文献   

6.
The effect of calcium and a soluble cytoplasmic activator on (Ca2+ + Mg2+)-ATPase of density-separated human red cells was investigated. At all calcium concentrations tested, dense (old) lysed cells and their isolated membranes displayed lower activities as compared to the light (young) cells and their membranes. Isolated membranes from all density red cell fractions showed two distinct (Ca2+ + Mg2+)-ATPase activities; one at low calcium and another at moderate calcium concentrations. At high calcium concentration, (Ca2+ + Mg2+)-ATPase activity of isolated membranes was low in all cell fractions. In contrast to the isolated membranes, lysed cells from all density fractions had a maximum (Ca2+ + Mg2+)-ATPase activity only at a low concentration of calcium, while moderate and high calcium concentrations produced low activity. Upon isolation of membranes, a substantial loss of (Ca2+ + Mg2+)-ATPase activity took place from all density cell fractions. Upon membrane isolation, the relative loss of (Ca2+ + Mg2+)-ATPase activity at low Ca2+ concentration was greater in older cells. The extent of stimulation of (Ca2+ + Mg2+)-ATPase by the activator at low calcium concentration was 3–4-fold greater in older cell membranes than in the young ones.These data suggest that the lower (Ca2+ + Mg2+)-ATPase activity in old cells could be accounted for by a selective loss of (Ca2+ + Mg2+)-ATPase activity at low Ca2+ concentration presumably due to reduced affinity of old cell membranes to activator protein.  相似文献   

7.
A membrane fraction enriched in axolemma was obtained from optic nerves of the squid (Sepiotheutis sepioidea) by differential centrifugation and density gradient fractionation. The preparation showed an oligomycin- and NaN3-insensitive (Ca2+ + Mg2+)-ATPase activity. The dependence of the ATPase activity on calcium concentration revealed the presence of two saturable components. One had a high affinity for calcium (K121 = 0.12 μM) and the second had a comparatively low affinity (K212 = 49.5 μM). Only the high-affinity component was specifically inhibited by vanadate (K1 = 35 μM). Calmodulin (12.5 μ/ml) stimulated the (Ca2+ + Mg2+)-ATPase by approx. 50%, and this stimulation was abolished by trifluoperazine (10 μM). Further treatment of the membrane fraction with 1% Nonidet P-40 resulted in a partial purification of the ATPase about 15-fold compared to the initial homogenate. This (Ca2+ + Mg2+)-ATPase from squid optic nerve displays some properties similar to those of the uncoupled Ca2+-pump described in internally dialyzed squid axons, suggesting that it could be its enzymatic basis.  相似文献   

8.
Organophosphorus insecticides parathion and methylparathion non-competitively inhibited the activity of (Ca2+ + Mg2+)-ATPase bound to and solubilized from pig erythrocyte membrane. Both enzyme preparations exhibited biphasic substrate curves displaying the existence of two functional active sites with low and high affinity to ATP. Also, the relationship between the activity of bound enzyme and Ca2+ concentration was biphasic. The activity reached maximum at 20 μM then dropped progressively as the Ca2+ concentration was raised. The inhibition of the activity was more pronounced for parathion than for methylparathion and the solubilized enzyme preparation was more affected than the bound one. The inhibition constants (Ki) for parathion for bound enzyme were 55 and 158 μM for high- and low-affinity active sites, respectively; for methylparathion these values equalled 74 and 263 μM, respectively. Ki values for parathion were 36 and 118 μM for solubilized enzyme (high- and low-affinity sites, respectively), for methylparathion −62 and 166 μM, respectively. The magnitude of the effect was greater for a low Ca2+ concentration, which could arise from different conformational states of the enzyme at different calcium concentrations. The results of the experiment suggest that the insecticides inhibited the ATPase by binding to a site on the enzyme rather than by the interaction with associated lipids, although lipids could weaken the action of the compounds due to the strong affinity of organophosphorus insecticides to lipids.  相似文献   

9.
Biochemical and kinetic properties under identical substrate and reaction conditions were obtained for an ATP-dependent Ca2+ pump and (Ca2+ + Mg2+)-ATPase in synaptosome membrane vesicles prepared from the brain of the moth, Mamestra configurata. Both the ATP-dependent Ca2+ pump and (Ca2+ + Mg2+)-ATPase had single, high-affinity binding sites for ATP (Km = 14 and 116 μM, respectively), Ca2+free (Km = 0.13 nM and 0.072 nM, respectively), and Mg2+ (Km = 1.1 mM and 0.07 mM, respectively). Both systems were relatively little affected by K+ and were insensitive to ouabain, an inhibitor of (Na+ + K+)-ATPase. The results indicate that the ATP-dependent Ca2+ pump and (Ca2+ + Mg2+)-ATPase are functionally coupled in synaptic membranes and constitute a mechanism for Ca2+ transport in the brain of M. configurata. Although moth brain (Ca2+ + Mg2+)-ATPase is maximally active at nanomolar concentrations of free calcium ion, the enzyme retains at least one-half of its maximal activity at micromolar calcium concentrations, indicating either that the enzyme has two binding sites for calcium (a high-affinity site at nanomolar Ca2+free and a low-affinity site at micromolar Ca2+free), or that there are two enzymes with high and low affinity for calcium, respectively. Calcium extrusion from brain neurones of M. configurata may operate in a two-stage, concentration-dependent process in which a first stage, low-affinity pump reduces intraneuronal calcium to a concentration at which a second stage, high-affinity pump becomes activated.  相似文献   

10.
The lipid requirement of the (Ca2+ + Mg2+)-stimulated ATPase of human erythrocytes has been studied. The enzyme activity was lost after removal of the phospholipids using phospholipase A2 from Naja naja and serum albumin. Optimal restoration of the (Ca2+ + Mg2+)-ATPase activity in the partially lipid-depleted membranes was obtained with oleate. The reactivation was not due to the removal of a permeability barrier for ATP, since lysolecithin or cholate did not show latent activity. Reactivation was also obtained with several negatively charged phospholipids. Among the ones normally found in the erythrocyte membranes, only phosphatidyl serine reactivated significantly.  相似文献   

11.
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   

12.
Effects of γ-aminobutyric acid (GABA) and ethanol on Mg2+-ATPase from mitochondrial and microsomal fractions of the fish brain were studied. GABA (10-8–10-4 M) activates microsomal Mg2+-ATPase, but has no effect on the mitochondrial enzyme activity. This effect of GABA on the microsomal Mg2+-ATPase was absent in the presence of 8% ethanol. Ethanol at 1– 10% concentrations inhibits the basal microsomal Mg2+-ATPase and has no effect on the mitochondria enzyme. Using cytochemical technique, Mg2+-ATPase was revealed both in neurons and in glial cells. The ethanol-sensitive Mg2+-ATPase is located in the area of synaptic junctions and is bound to plasma, vesicular, and smooth endoplasmic reticulum membranes.  相似文献   

13.
14.
H. Liß  E. W. Weiler 《Planta》1994,194(2):169-180
Procedures have been developed which allow the preparation of highly pure endoplasmic reticulum and plasma membrane from tendrils ofBryonia dioica. These and further membrane fractions were used to study vanadate-sensitive ATPase activity as well as Mg2+ATP-driven transport of45Ca2+. Calcium-translocating ATPases were detected in the endoplasmic reticulum, the plasma membrane and the mitochondrial fraction and characterized kinetically and with respect to the effects of various inhibitors. The endoplasmic-reticulum Ca2+-translocating ATPase was stimulated by KCl and was calmodulin-dependent. The plasma-membrane enzyme was not affected by these agents. These, as well as the inhibitor data, show that the Ca2+-translocating ATPases of the endoplasmic reticulum and the plasma membrane are distinctly different enzymes. Upon mechanical stimulation, the activities of the vanadate-sensitive K+, Mg2+-ATPase and the Ca2+-translocating ATPase(s) increased rapidly and transiently, indicating that increasing transmembrane proton and calcium fluxes are involved in the early stages of tendril coiling.Abbreviations CAM calmodulin - CCCP carbonylcyanidem-chlorophenylhydrazone - IC50 concentration giving 50% inhibition - PM plasma membrane - rER rough endoplasmic reticulum - sER smooth endoplasmic reticulum - FC fusicoccin - U3+U3 the two PM-rich upper phases obtained after phase partitioning of microsomal membranes The authors wish to thank the Deutsche Forschungsgemeinschaft, Bonn, Germany, and the Fonds der Chemischen Industrie, Frankfurt, Germany (literature provision) for financial support.  相似文献   

15.
Demonstration of a high affinity Ca2+ ATPase in rat liver plasma membranes   总被引:4,自引:0,他引:4  
Rat liver plasma membranes contained a high affinity Ca2+-ATPase which had an apparent half saturation constant of 0.2 μM for calcium. The Ca2+-ATPase was not stimulated by adding magnesium and/or calmodulin. Conversely, the addition of these substances diminished the calcium-stimulation of the ATPase. Orthovanadate (7 nM-2 mM), mitochondrial ATPase blockers (NaN3, KCN, dicyclohexylcarbodiimide), Na+, K+ and ouabain had no effect on the ATPase activity. The ATPase was separated from nonspecific divalent cation stimulatable ATPase (Mg2+-ATPase) by solubilization with Triton X-100 followed by a Sephadex G-200 column chromatography and showed an apparent molecular weight of 200,000.  相似文献   

16.
(1) The effects of calmodulin binding on the rates of Ca2+-dependent phosphorylation and dephosphorylation of the red-cell Ca2+ pump, have been tested in membranes stripped of endogenous calmodulin or recombined with purified calmodulin. (2) In Mg2+-containing media, phosphorylation and dephosphorylation rates are accelerated by a large factor (at 0°C), but the steady-state level of phosphoenzyme is unaffected by calmodulin binding (at 0°C and 37°C). In Mg2+-free media, slower rates of phosphoenzyme formation and hydrolysis are observed, but both rates and the steady-state phosphoenzyme level are raised following calmodulin binding. (3) At 37°C and 0°C, the rate of (Ca2+ + Mg2+)-ATPase activity is stimulated maximally by 6–7-fold, following calmodulin binding. At 37°C the apparent Ca2+ affinity for sustaining ATP hydrolysis is raised at least 20-fold, Km(Ca) ? 10 μM (—calmodulin) and Km(Ca) < 0.5 μM (+ calmodulin), but at 0°C the apparent Ca2+ affinity is very high in calmodulin-stripped membranes and little or no effect of calmodulin is observed (Km(Ca) ? 3–4 · 10-8 M). (Ca2+ + Mg2+)-ATPase activity in calmodulin activated membranes and at saturating ATP levels, is sharply inhibited by addition of calcium in the range 50–2000 μM. (4) A systematic study of the effects of the nucleotide species MgATP, CaATP and free ATP on (Ca2+ + Mg2+)-ATPase activity in calmodulin-activated membranes reveals: (a) In the 1–10 μmolar concentration range MgATP, CaATP and free ATP appear to sustain (Ca2+ + Mg2+)-ATPase activity equally effectively. (b) In the range 100–2000 μM, MgATP accelerates ATP hydrolysis (Km(MgATP) ? 360 μM), and CaATP is an inhibitor (Ki(CaATP) ? 165 μM), probably competing with MgATP fo the regulatory site. (5) The results suggest that calmodulin binding alters the conformational state of the Ca2+- pump active site, producing a high (Ca2+ + Mg2+)-ATPase activity, high Ca2+ affinity and regulation of activity by MgATP.  相似文献   

17.
Fedirko  N.  Vats  Yu.  Kruglikov  I.  Voitenko  N. 《Neurophysiology》2004,36(3):169-173
In a rat model of streptozotocin (STZ)-induced diabetes, we earlier showed that under these conditions the concentration of free cytosolic Ca2+ in input neurons of the nociceptive system increases, Ca2+ signals are prolonged, while Ca2+ release from intracellular calcium stores decreases. The aim of our study was to test the hypothesis that changes in the activities of Ca2+,Mg2+-ATPases of the endoplasmic reticulum (SERCA) and plasmalemma (PMCA) could be responsible for diabetes-induced disorders of calcium homeostasis in nociceptive neurons. We measured the Ca2+,Mg2+-ATPase activities in microsomal fractions obtained from tissues of the dorsal root ganglia (DRG) and spinal dorsal horn (DH) of control rats and rats with experimentally induced diabetes. The integral specific Ca2+,Mg2+-ATPase activity in microsomes from diabetic rats was lower than that in the control group. The activity of SERCA in samples of DRG and DH of diabetic rats was reduced by 50 ± 8 and 48 ± 12%, respectively, as compared with the control (P < 0.01). At the same time, the activity of PMCA decreased by 63 ± 6% in DRG and by 60 ± 9% in DH samples (P < 0.01). We conclude that diabetic polyneuropathy is associated with the reduction of the rate of recovery of the Ca2+ level in the cytosol of DRG and DH neurons due to down-regulation of the SERCA and PMCA activities.  相似文献   

18.
Calcium is actively transported into intracellular organelles and out of the cytoplasm by Ca2+/Mg2+-ATPases located in the endoplasmic reticulum and plasma membranes. We studied the effects of aluminum on calcium transport in the adult rat brain. We examined 45Ca-uptake in microsomes and Ca2+-ATPase activity in microsomes and synaptosomes isolated from the frontal cortex and cerebellum of adult male Long-Evans rats. ATP-dependent45Ca-uptake was similar in microsomes from both brain regions. The addition of 50-800 μM AICI3 resulted in a concentration-dependent inhibition of 45Ca-uptake. Mg2+-dependent Ca2+-ATPase activity was significantly lower in synaptosomes compared to microsomes in both frontal cortex and cerebellum. In contrast to the uptake studies, AICI3 stimulated Mg2+-dependent Ca2+-ATPase activity in both microsomes and synaptosomes from both brain regions. To determine the relationship between aluminum and Mg2+, we measured ATPase activity in the presence of increasing concentrations of Mg2+ or AICI3. Maximal ATPase activity was obtained between 3 and 6 mM Mg2+. When we substituted AICI3 for Mg2+, ATPase activity was also stimulated in a concentration-dependent manner, but to a greater extent than with Mg2+. One interpretation of these data is that aluminum acts at multiple sites to displace both Mg2+ and Ca2+, increasing the activity of the Ca2+-ATPase, but disrupting transport of calcium.  相似文献   

19.
20.
Erythrocyte membranes prepared by three different procedures showed (Mg2+ + Ca2+)-ATPase activities differing in specific activity and in affinity for Ca2+. The (Mg2+ + Ca2+)-ATPase activity of the three preparations was stimulated to different extents by a Ca2+-dependent protein activator isolated from hemolystes. The Ca2+ affinity of the two most active preparations was decreased as the ATP concentration in the assay medium was increased. Lowering the ATP concentration from 2 mM to 2–200 μM or lowering the Mg:ATP ratio to less than one shifted the (Mg2+ + Ca2+)-ATPase activity in stepwise hemolysis membranes from mixed “high” and “low” affinity to a single high Ca2+ affinity. Membranes from which soluble proteins were extracted by EDTA (0.1 mM) in low ionic strengh, or membranes prepared by the EDTA (1–10 mM) procedure, did not undergo the shift in the Ca2+ affinity with changes in ATP and MgCl2 concentrations. The EDTA-wash membranes were only weakly activated by the protein activator. It is suggested that the differences in properties of the (Mg2+ + Ca2+)-ATPase prepared by these three procedures reflect differences determined in part by the degree of association of the membrane with a soluble protein activator and changes in the state of the enzyme to a less activatable form.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号