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1.
We have studied the activities of Ca2+-stimulated ATPase in rat heart sarcolemma upon modulating the redox state of membrane thiol groups with dithiothreitol (DTT). The suitability of alamethicin to unmask the latent activity of this enzyme was also investigated. The Ca2+-stimulated ATPase in sarcolemma exhibited two activation sites — one with low affinity (Km = 0.70 ± 0.2 mM; Vmax = 10.0 ± 2.2 mol Pi/mg/h) and the other with high affinity (Km = 0.16 ± 0.7 mM; Vmax = 4.6 ± 0.8 mol Pi/mg/h) for Mg2+ATP. Alamethicin at a ratio of 1:1 with the sarcolemmal protein caused a 3-fold activation of Ca2+-stimulated ATPase without affecting its sensitivity to Ca2+ or Mg2+ATP. Treatment of sarcolemma with deoxycholate or sodium dodecyl sulfate resulted in a total loss of the enzyme activity; high concentrations of alamethicin also showed a detergent-like action on the sarcolemmal vesicles. DTT at 5–10 mM concentrations caused a 4–5 fold activation of Ca2+-stimulated ATPase in sarcolemma and this effect was observed to be dependent on the concentration of Mg2+ATP. DTT increased the affinity of the enzyme to Mg2+ATP at the high affinity site and enhanced the Vmax at the low affinity site in addition to increasing the sensitivity of Ca2+-stimulated ATPase to Ca2+. DTT protected the Ca2+-stimulated ATPase against deterioration by detergents and restored the enzyme activity after treatment with N-ethylmaleimide. The mechanism of action of DTT on Ca2+-stimulated ATPase may involve the reduction of essential thiols at the active site of the enzyme or its interaction with specific DTT-dependent inhibitor protein. No changes in the sensitivity of sarcolemmal Ca2+-stimulated ATPase to orthovanadate was evident in the absence or presence of DTT and alamethicin. The results suggest the use of both DTT and alamethicin for the determination of Ca2+-stimulated ATPase activity in sarcolemmal preparations. 相似文献
2.
A high affinity Ca2+/Mg2+ ATPase has been identified and localized in synaptic membrane subfractions. This enzyme is stimulated by low concentrations of Ca2+ (1 M) believed to approximate the range of Ca2+ in the synaptosomal cytosol (0.1 to 5.0 M). The opiate agonist levorphanol, in a concentration-dependent fashion, inhibited Ca2+-stimulated ATP hydrolysis in lysed synaptic membranes. This inhibition was reversed by naloxone, while dextrorphan, the inactive opiate isomer, was without effect. Inhibition by levorphanol was most pronounced in a subfraction of synaptic membranes (SPM-1). The inhibition of Ca2+-stimulated ATP hydrolysis was characterized by a reduction inV
max for Ca2+. Levorphanol pretreatment reduced the Hill coefficient (HN) of 1.5 to 0.7, suggesting cooperative interaction between the opiate receptor and the enzyme protein. Levorphanol, but not dextrorphan, also inhibited (28%) ATP-dependent Ca2+ uptake by synaptic membranes. Opiate ligand stereoisomers were tested for their effects on calmodulin stimulating of high affinity Ca2+/Mg2+ ATPase in synaptic membranes. Levorphanol (10 M), but not the inactive stereoisomer (+)dextrorphan, significantly inhibited (35%) the calmodulin-activated Ca2+-dependent ATP hydrolysis activity in a preparation of lysed synaptic membranes. Both Ca2+-dependent and calmodulin-dependent stimulation of the enzyme in the presence of optimal concentrations of the other co-substrate were inhibited by levorphanol (35–40%) but not dextrorphan. Inhibition of ATP hydrolysis was characterized by a reduction inV
max for both Ca2+ and calmodulin stimulation of the enzyme. Calmodulin stimulation of enzyme activity was most pronounced in SPM-1, the membrane fraction which also exhibits the maximal opiate inhibition (40%) of the Ca2+-ATPase. The results demonstrate that opiate receptor activation inhibits a high affinity Ca2+/Mg2+ ATPase in synaptic plasma membranes in a stereospecific fashion. The inhibition of the enzyme may occur by a mechanism involving both Ca2+ and calmodulin. Inhibition of calmodulin activation may contribute to the mechanism by which opiate ligands disrupt synaptosomal Ca2+ buffering mechanisms. Changes in the cytosolic distribution of synaptosomal Ca2+ following inhibition of Ca2+/Mg2+ ATPase may underlie some of the pharmacological effects of opiate drugs. 相似文献
3.
Energy-dependent endocytosis and the low Ca2+ affinity Ca2+-stimulated ATPase activity of erythrocyte ghosts were inhibited concurrently by two inhibitors, carbonylcyanide-m-chlorophenylhydrazone (CCCP) and N-naphthylmaleimide. The conditions required to observe 50% inhibition of this Ca2+-stimulated ATPase activity with either inhibitor were the same conditions required to observe this level of inhibition of endocytosis. Under these conditions, none of the other ATPase activities measured were inhibited more than 20% by either of these reagents. This concurrence of inhibition of endocytosis and the low-affinity Ca2+-stimulated ATPase and the possible involvement of this ATPase in the mechanism by which endocytosis occurs is discussed. 相似文献
4.
Carlos Humberto Pedemonte Héctor Federico Balegno 《Molecular and cellular biochemistry》1981,40(2):105-1071
Summary The effect of Ca2+ on the stability of the Ca2+-stimulated ATPase has been investigated. Our results showed that the preincubation of the rat red cell membranes in presence of Ca2+ causes an irreversible inhibition of the enzyme. The same effect was obtained with Ba2+ instead of Ca2+. Once initiated the inactivation of the enzyme could be halted by the addition of ethylene glycol bis (B-amino ethyl ether) N,N-tetra acitic acid (EGTA), but inactivation was irreversible. The presence of ATP in the preincubation with Ca2+ prevented the inactivation but calmodulin did not. 相似文献
5.
The effects of ethanol in vitro on calmodulin-dependent Ca2+-activated ATPase (CaM–Ca2+-ATPase) activity were studied in synaptic plasma membranes (SPM) prepared from the brain of normal and chronically ethanol-treated rats. In SPM from normal animals, ethanol at 50–200 mM inhibited the Ca2+-ATPase activity. Lineweaver-Burk analysis indicates that the inhibition was the result of a decreased affinity of the enzyme for calmodulin, whereas the maximum activity of the enzyme was not changed. Arrhenius analysis indicates that the enzyme activity was influenced by lipid transition of the membranes, and ethanol in vitro resulted in a shift of the transition temperature toward a lower value. From animals receiving chronic ethanol treatment (3 weeks), the SPM were resistant to the inhibitory effect of ethanol on the enzyme activity. The resistance to ethanol inhibition was correlated with a higher enzyme affinity for calmodulin and a higher transition temperature, as compared with normal SPM. Since the calmodulin-dependent Ca2+-ATPase in synaptic plasma membranes is believed to be the Ca2+ pump controlling free Ca2+ levels in synaptic terminals, its inhibition by ethanol could therefore lead to altered synaptic activity.Abbreviations used ATPase
adenosine triphosphatase
- CaM
calmodulin
- CaM–Ca2+-ATPase
calmodulin-dependent Ca2+-activated ATPase
- EGTA
ethylene-bis(oxyethylenenitrilo)tetraacetic acid
- EtOH
ethanol
- Hepes
N—2-hydroxyethylpiperazine-N-2-ethanesulfonic acid
- SPM
synaptic plasma membranes
- TFP
trifluoperazine
- Tris
tris(hydroxymethyl)aminomethane
- Km
Michaelis constant
- Td
transition temperature
- Vmax
maximum velocity 相似文献
6.
《Comparative biochemistry and physiology. B, Comparative biochemistry》1991,98(4):753-758
- 1.1. In the plasma membrane of mussel gill cells an ouabain insensitive, Ca2+-activated ATPase activity is present. The ATPase has high Ca2+ affinity (Kma = 0.3 μM).
- 2.2. The optimum assay conditions to evaluate the enzymatic activity of the Ca2+-stimulated ATPase at 19°C are: 120–300 mM KCl ionic strength, pH 7.0 and 2 mM ATP. As for mammalian enzymes, the Ca2+ ATPase activity is stimulated by DTT (0.5–1 mM) and it is inhibited by low concentrations of vanadate (10–50 μM) and -SH inhibitors such as PCMB and PCMBS (10 μM); the enzyme appears to be calmodulin insensitive.
- 3.3. Electrophoretic analyses of plasma membrane proteins demonstrate that: (a) Ca2+ at n-μM concentrations is necessary to activate ATP hydrolysis with consequent formation of the enzyme-phosphate complex; (b) the steady state concentration of the phosphorylated intermediate is increased in the presence of La3+; (c) the mol. wt of Ca2+ ATPase is about 140 kDa.
- 4.4. Low Ca2+ concentrations (n-μM) are sufficient to stimulate the ATP-dependent Ca2+ uptake by plasma membrane inside-out vesicles.
- 5.5. The results indicate that the Ca2+ pump present in the gill plasma membranes could be responsible for Ca2+ extrusion and therefore involved in maintaining the cytosolic Ca2+ concentration within physiological levels.
7.
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
8.
Enn K. Seppet Frantisek Kolar Ian M. C. Dixon Tomoji Hata Naranjan S. Dhalla 《Molecular and cellular biochemistry》1993,129(2):145-159
In order to examine the regulatory role of thyroid hormone on sarcolemmal Ca2+-channels, Na+–Ca2+ exchange and Ca2+-pump as well as heart function, the effects of hypothyroidism and hyperthyroidism on rat heart performance and sarcolemmal Ca2+-handling were studied. Hyperthyroid rats showed higher values for heart rate (HR), maximal rates of ventricular pressure development+(dP/dt)max and pressure fall–(dP/dt)max, but shorter time to peak ventricular pressure (TPVP) and contraction time (CT) when compared with euthyroid rats. The left ventricular systolic pressure (LVSP) and left ventricular end-diastolic pressure (LVEDP), as well as aortic systolic and diastolic pressures (ASP and ADP, respectively) were not significantly altered. Hypothyroid rats exhibited decreased values of LVSP, HR, ASP, ADP, +(dP/dt)max and –(dP/dt)max but higher CT when compared with euthyroid rats; the values of LVEDP and TPVP were not changed. Studies with isolated-perfused hearts showed that while hypothyroidism did not modulate the inotropic response to extracellular Ca2+ and Ca2+ channel blocker verapamil, hyperthyroidism increased sensitivity to Ca2+ and decreased sensitivity to verapamil in comparison to euthyroid hearts. Studies of [3H]-nitrendipine binding with purified cardiac sarcolemmal membrane revealed decreased number of high affinity binding sites (Bmax) without any change in the dissociation constant for receptor-ligand complex (Kd) in the hyperthyroid group when compared with euthyroid sarcolemma; hypothyroidism had no effect on these parameters. The activities of sarcolemmal Ca2+-stimulated ATPase, ATP-dependent Ca2+ uptake and ouabain-sensitive Na+–K+ ATPase were decreased whereas the Mg2+-ATPase activity was increased in hypothyroid hearts. On the other hand, sarcolemmal membranes from hyperthyroid samples exhibited increased ouabain-sensitive Na+–K+ ATPase activity, whereas Ca2+-stimulated ATPase, ATP-dependent Ca2+ uptake, and Mg2+-ATPase activities were unchanged. The Vmax and Ka for Ca2+ of cardiac sarcolemmal Na+–Ca2+ exchange were not altered in both hyperthyroid and hypothyroid states. These results indicate that the status of sarcolemmal Ca2+-transport processes is regulated by thyroid hormones and the modification of Ca2+-fluxes across the sarcolemmal membrane may play a crucial role in the development of thyroid state-dependent contractile changes in the heart. 相似文献
9.
《Comp. Biochem. Physiol. C, Comp. Pharmacol. Toxicol.》1995,110(2):119-125
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
10.
Mg2+ and ATP effects on K+ activation of the Ca2+-transport ATpase of cardiac sarcoplasmic reticulum
Larry R. Jones 《生物化学与生物物理学报:生物膜》1979,557(1):230-242
ATP and the divalent cations Mg2+ and Ca2+ regulated K+ stimulation of the Ca2+-transport ATPase of cardiac sarcoplasmic reticulum vesicles. Millimolar concentrations of total ATP increased the K+-stimulated ATPase activity of the Ca2+ pump by two mechanisms. First, ATP chelated free Mg2+ and, at low ionized Mg2+ concentrations, K+ was shown to be a potent activator of ATP hydrolysis. In the absence of K+ ionized Mg2+ activated the enzyme half-maximally at approximately 1 mM, whereas in the presence of K+ the concentration of ionized Mg2+ required for half-maximal activation was reduced at least 20-fold. Second MgATP apparently interacted directly with the enzyme at a low affinity nucleotide site to facilitate K+-stimulation. With a saturating concentration of ionized Mg2+, stimulation by K+ was 2-fold, but only when the MgATP concentration was greater than 2 mM. Hill plots showed that K+ increased the concentration of MgATP required for half-maximal enzymic activation approx. 3-fold.Activation of K+-stimulated ATPase activity by Ca2+ was maximal at anionized Ca2+ concentration of approx. 1 μM. At very high concentrations of either Ca2+ or Mg2+, basal Ca2+-dependent ATPase activity persisted, but the enzymic response to K+ was completely inhibited. The results provide further evidence that the Ca2+-transport ATPase of cardiac sarcoplasmic reticulum has distinct sites for monovalent cations, which in turn interact allosterically with other regulatory sites on the enzyme. 相似文献
11.
An endogenous soluble protease has been demonstrated to unmask a Ca2+-stimulated ATPase activity in purified dog gastric microsomes. The presence of ATP during protease treatment appears essential for the manifestation of the gastric Ca2+-stimulated ATPase activity. The endogenous protease appears to have trypsin-like activity, since soybean trypsin inhibitor completely blocks the protease effect. Manifestation of the Ca2+-stimulated ATPase occurs without affecting the microsomal (H+ +K+)-ATPase activity and associated H+ uptake ability. The unmasked Ca2+-stimulated ATPase appears insensitive to calmodulin. Possible roles of the enzyme in the regulation of gastric H+ transport have been discussed. 相似文献
12.
A fraction was isolated from human red cells capable of decreasing Ca2+-stimulated with a parallel increase of Mg2+-stimulated ATPase. This fraction seems to produce an increase of the apparent affinity for Ca2+. But, when the fraction concentration was increased, there was a decrement of Ca2+-stimulated ATPase up to the point of complete abolishment. At this point, Mg2+-stimulated ATPase reached the initial level of the Ca2+-stimulated ATPase. 相似文献
13.
Summary During the first four mitotic division cycles of Lymnaea stagnalis embryos, we have detected cell cycle-dependent changes in the pattern of transcellular ionic currents and membrane-bound Ca2+-stimulated ATPase activity. Ionic currents ranging from 0.05 to 2.50 A/cm2 have been measured using the vibrating probe technique. Enzyme activity was detected using Ando's cytochemical method (Ando et al. 1981) which reveals Ca2+/Mg2+ ATPase localization at the ultrastructural level, and under high-stringency conditions with respect to calcium availability, it reveals Ca2+-stimulated ATPase. The ionic currents and Ca2+-stimulated ATPase localization have in common that important changes occur during the M-phase of the cell cycles. Minimal outward current at the vegetal pole coincides with metaphase/anaphase. Maximal inward current at the animal pole coincides with the onset of cytokinesis at that pole. Ca2+-stimulated ATPase is absent from one half of the embryo at metaphase/anaphase of the two- and four-cell stage, whereas it is present in all cells during the remaining part of the cell cycle. Since fluctuations of cytosolic free calcium concentrations appear to correlate with both karyokinesis and cytokinesis, we speculate that part of the cyclic pattern of Ca2+-stimulated ATPase localization and of the transcellular ionic currents reflects the elevation of cytosolic free calcium concentration during the M-phase.
Offprint requests to: D. Zivkovic 相似文献
14.
Kevin K. W. Wang Basil D. Roufogalis Antonio Villalobo 《The Journal of membrane biology》1989,112(3):233-245
Summary Calpain I purified from human erythrocyte cytosol activates both the ATP hydrolytic activity and the ATP-dependent Ca2+ transport function of the Ca2+-translocating ATPase solubilized and purified from the plasma membrane of human erythrocytes and reconstituted into phosphatidylcholine vesicles. Following partial proteolysis of the enzyme by calpain I, both the initial rates of calcium ion uptake and ATP hydrolysis were increased to near maximal levels similar to those obtained upon addition of calmodulin. The proteolytic activation resulted in the loss of further stimulation of the rates of Ca2+ translocation or ATP hydrolysis by calmodulin as well as an increase of the affinity of the enzyme for calcium ion. However, the mechanistic Ca2+/ATP stoichiometric ratio was not affected by the proteolytic treatment of the reconstituted Ca2+-translocating ATPase. The proteolytic activation of the ATP hydrolytic activity of the reconstituted enzyme could be largely prevented by calmodulin. Different patterns of proteolysis were obtained in the absence or in the presence of calmodulin during calpain treatment: the 136-kDa enzyme was transformed mainly into a 124-kDa active ATPase fragment in the absence of calmodulin, whereas a 127-kDa active ATPase fragment was formed in the presence of calmodulin. This study shows that calpain I irreversibly activates the Ca2+ translocation function of the Ca2+-ATPase in reconstituted proteoliposomes by producing a calmodulin-independent active enzyme fragment, while calmodulin antagonizes this activating effect by protecting the calmodulin-binding domain against proteolytic cleavage by calpain. 相似文献
15.
Anil K. Verma John T. Penniston Schmuel Muallem Virgilio Lew 《Journal of bioenergetics and biomembranes》1984,16(5-6):365-378
Antibodies raised in rabbits against the purified erythrocyte membrane Ca2+ pumping ATPase were affinity-purified using an ATPase-Sepharose column. Addition of a few molecules of the purified antibody per molecule of ATPase was sufficient to inhibit the ATPase activity. Extensively washed ghosts or preincubated pure ATPase sometimes develop an appreciable Mg2+-ATPase activity. In such cases, the antibodies inhibited the Mg2+-ATPase as well as the Ca2+-ATPase. This is consistent with the hypothesis that a portion of the Mg2+-ATPase activity of ghosts is derived from the Ca2+-ATPase. When nitrophenylphosphatase activity was observed, both Mg2+ - and Ca2+-stimulated activities were observed. Only the Ca2+ activity was inhibited by the antibodies, confirming that this activity is due to the Ca2+ pump, and suggesting that the Mg2+-nitrophenylphosphatase is due to a separate enzyme. Amounts of antibody comparable to those which inhibited the Ca2+-ATPases had no effect on the Na+-K+-ATPase; 4-fold higher amounts of antibody significantly stimulated the Na+-K+-ATPase, but this effect of the antibody was not specific: Immunoglobulins from the nonimmune serum also significantly stimulated the Na+-K+-ATPase.In resealed erythrocyte membranes, antibodies incorporated into the ghosts inactivated the Ca2+-ATPase, while antibodies added to the outside had no significant effect. 相似文献
16.
《Biochemical and biophysical research communications》1985,126(3):1196-1200
The Ca2+-dependent ATPase activity of sarcoplasmic reticulum was inhibited when membrane vesicles were incubated at 0°C in presence of thiols. 2-mercaptoethanol was the most effective inhibitor from the thiols tested. The effect of 2-mercaptoethanol on the ATPase activity was biphasic; enzyme inhibition originally increased and then decreased with increasing thiol concentration. The inhibitory action of this thiol was significantly higher at low membrane concentrations and the rate of inactivation at 22°C was considerably lower than that at 0°C. Ca2+-ATPase previously inhibited by 2-mercaptoethanol was partially reactivated by incubation with periodate. 相似文献
17.
The extent of the negative cooperativity with MgATP of the Ca2+-stimulated ATPase activity of sarcoplasmic reticulum has been studied with various membrane preparations and under various conditions. Preparations studied were fragmented sarcoplasmic reticulum vesicles, deoxycholate-solubilized and fractionated ATPase, triton extracted reticulum, vesicles reconstituted from either detergent, and limited trypsin digests of the reticulum. Conditions studied were suboptimal, optimal, and inhibitory Ca2+ concentrations; temperatures from 13 to 46 °C; 1 or 5 mm MgCl2; 0.1 m KCl, 0.1 m NaCl, or no added salt; and Triton or deoxycholate present in the assay. With preparations in which vesicles could accumulate Ca2+ ion, the ionophore A23187 was added to prevent inhibition by internal Ca2+ ions. Under all circumstances, the negative cooperativity of MgATP was present (Hill coefficient of 0.2 to 0.8), indicating the persistence of the properties of the enzyme molecule and its lipid environment giving rise to kinetic negative cooperativity. Attempts to measure the number of ATP sites by protection against N-ethylmaleimide inactivation and by binding of an analog suggested, but did not prove, that there was only one specific, active ATP binding site below 0.5 mm. These results are interpreted to be consistent with either of two mechanisms for ATP cooperativity of the Ca2+-stimulated ATPase activity of sarcoplasmic reticulum: (a) a single, high affinity ATP active site and a second, lower affinity “allosteric” activator site; or (b) a single ATP site which demonstrates two affinities through some kinetic mechanism such as a substrate-induced, slow transition. 相似文献
18.
S G O'Neal D B Rhoads E Racker 《Biochemical and biophysical research communications》1979,89(3):845-850
Vanadate is a potent inhibitor of the Ca2+-ATPase activity of sarcoplasmic reticulum in the presence of A-23187. The purified enzyme is sensitive to vanadate even in the absence of the ionophore. Ca2+ and norepinephrine protect the enzyme against inhibition of vanadate. The nonspecificity of vanadate is emphasized by the finding of inhibition of several other ATPases including the Ca2+Mg2+-ATPases of the ascites and human red cell plasma membranes, Mg2+-ATPase of the ascites plasma membrane, and the K+-ATPases of and hog gastric mucosal cell membranes. The ascites plasma membrane Ca2+-ATPase (an ecto ATPase) and mitochondrial ATPase are not inhibited by vanadate. 相似文献
19.
Betty L. Black Jay M. McDonald Leonard Jarett 《Archives of biochemistry and biophysics》1980,199(1):92-102
The presence of an energy-dependent calcium uptake system in adipocyte endoplasmic reticulum (D. E. Bruns, J. M. McDonald, and L. Jarett, 1976, J. Biol. Chem.251, 7191–7197) suggested that this organelle might possess a calcium-stimulated transport ATPase. This report describes two types of ATPase activity in isolated microsomal vesicles: a nonspecific, divalent cation-stimulated ATPase (Mg2+-ATPase) of high specific activity, and a specific, calcium-dependent ATPase (Ca2+ + Mg2+-ATPase) of relatively low activity. Mg2+-ATPase activity was present in preparations of mitochondria and plasma membranes as well as microsomes, whereas the (Ca2+ + Mg2+)-ATPase activity appeared to be localized in the endoplasmic reticulum component of the microsomal fraction. Characterization of microsomal Mg2+-ATPase activity revealed apparent Km values of 115 μm for ATP, 333 μm for magnesium, and 200 μm for calcium. Maximum Mg2+-ATPase activity was obtained with no added calcium and 1 mm magnesium. Potassium was found to inhibit Mg2+-ATPase activity at concentrations greater than 100 mm. The energy of activation was calculated from Arrhenius plots to be 8.6 kcal/mol. Maximum activity of microsomal (Ca2+ + Mg2+)-ATPase was 13.7 nmol 32P/mg/min, which represented only 7% of the total ATPase activity. The enzyme was partially purified by treatment of the microsomes with 0.09% deoxycholic acid in 0.15 m KCl which increased the specific activity to 37.7 nmol 32P/mg/min. Characterization of (Ca2+ + Mg2+)-ATPase activity in this preparation revealed a biphasic dependence on ATP with a Hill coefficient of 0.80. The apparent Kms for magnesium and calcium were 125 and 0.6–1.2 μm, respectively. (Ca2+ + Mg2+)-ATPase activity was stimulated by potassium with an apparent Km of 10 mm and maximum activity reached at 100 mm potassium. The energy of activation was 21.5 kcal/mol. The kinetics and ionic requirements of (Ca2+ + Mg2+)-ATPase are similar to those of the (Ca2+ + Mg2+)-ATPase in sarcoplasmic reticulum. These results suggest that the (Ca2+ + Mg2+)-ATPase of adipocyte endoplasmic reticulum functions as a calcium transport enzyme. 相似文献
20.
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