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1.
Purified red blood cell Ca2+-pump ATPase: evidence for direct inhibition by presumed anti-calmodulin drugs in the absence of calmodulin 总被引:2,自引:0,他引:2
A variety of presumed anti-calmodulin (anti-CaM) drugs was tested for their potential inhibitory effects on the isolated, purified and reconstituted Ca2+-pump ATPase of human red blood cell membranes. Anti-CaM drugs inhibited the Ca2+-pump ATPase both in the absence and presence of added CaM. Qualitatively similar inhibition was observed in two different ATPase assay systems. In asolectin vesicles in the absence of added CaM trifluoperazine (TFP), N-(6-aminohexyl)-5-chloro-1-naphthalene- sulfonamide (W-7), vinblastine, dibucaine, imipramine, propranolol and dimethylpropranolol (UM-272) were all inhibitory. Potency of anti-CaM drugs was generally greater on the enzyme reconstituted in asolectin vesicles than on the enzyme reconstituted in phosphatidylcholine vesicles, either in the presence or absence of CaM. The results emphasize that anti-CaM drugs have actions other than to bind to CaM. Possible direct interaction of amphipathic cationic anti-CaM drugs with the Ca2+-pump ATPase and/or its lipid environment is suggested. 相似文献
2.
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
3.
Summary Human erythrocyte Ca2+-translocating ATPase was solubilized from calmodulin-depleted membranes using the detergent Triton X-100, and subsequently purified by calmodulin-affinity chromatography. The purified enzyme was reconstituted in artificial phospholipid vesicles using a cholate-dialysis method and various phospholipids. The reconstituted enzyme was able to translocate Ca2+ inside the vesicles, both in the absence and in the presence of the Ca2+-chelating agent, oxalate, inside the vesicles. The tightness of coupling between ATP hydrolysis and cation translocation was investigated by the use of different ionophoretic compounds. The efficiency of Ca2+ translocation was measured by the ability of the ionophores to stimulate ATP hydrolytic activity of the reconstituted enzyme. It was found that the maximum stimulation of the ATP hydrolytic activity was induced by the electroneutral Ca2+/2H+ ionophore A23187 (9 to 10-fold). A Ca2+ ionophore unable to translocate H+, CYCLEX-2E, was less efficient in stimulating the activity of the reconstituted enzyme (two- to threefold). However, the combined addition of CYCLEX-2E plus protonophores further increased the ATP hydrolytic activity (around fourfold), whereas, the protonophores did not further stimulate ATP hydrolysis in the presence of A23187. Furthermore, in the absence of Ca2+ ionophore, the electroneutral K+(Na+)/H+ ionophoretic exchanger, nigericin, or the electroneutral Na+(K+)/H+ ionophoretic exchanger, monensin, stimulated the rate of ATP hydrolysis in the reconstituted enzyme two- or threefold, respectively. These results suggest that the Ca2+-ATPase not only translocates Ca2+ but also H+ in the opposite direction. 相似文献
4.
Frank F. Vincenzi 《Cell calcium》1981,2(4):387-409
Calmodulin (CaM) is a major intracellular receptor for Ca2+. CaM is thus a crucial receptor to consider in pharmacological modification of cellular activity. Potential mechanisms by which drugs may modify CaM effectiveness are considered in the context of its interaction with Ca2+ and in turn with its various effectors. Some examples of established drug mechanisms are considered. A wide range of chemical compounds representing diverse pharmacological classes are anti-CaM under some conditions. No simple relationships have been established between molecular level events and therapeutic applicability of anti-CaM compounds. 相似文献
5.
Reconstitution of the purified calmodulin-dependent (Ca2+ + Mg2+)-ATPase from smooth muscle 总被引:4,自引:0,他引:4
The purified calmodulin dependent (Ca2+ + Mg2+)-ATPase (CaMg ATPase) from porcine antral smooth muscle transports Ca2+ after reconstitution in lipid vesicles indicating that this enzyme is indeed a Ca2+-transport ATPase. For CaMg ATPase reconstituted in asolectin vesicles a good correlation was found between the time course of Ca2+ accumulation and the corresponding changes in CaMg ATPase activity. The ATPase activity was stimulated 8-fold by A23187, which further indicates a tight coupling between ATP hydrolysis and Ca2+ transport. Asolectin vesicles with incorporated enzyme accumulated Ca2+ with a ratio approaching one Ca2+ ion transported for each ATP hydrolyzed. For CaMg ATPase reconstituted in phosphatidylcholine vesicles on the other hand, Ca2+ transport and CaMg ATPase were poorly coupled as is shown by the approximately 3.5 fold stimulation by A23187. The activity of the CaMg ATPase when reconstituted in asolectin vesicles was stimulated 1.25 fold by calmodulin while in phosphatidylcholine a value of 4.25 was obtained. The CaMg ATPase activity of the enzyme reconstituted either in asolectin or phosphatidylcholine was, after its stimulation by A23187, still further stimulated by detergent by a factor of 5. 相似文献
6.
Ca 2+ uptake in reconstituted sarcoplasmic reticulum vesicles 总被引:3,自引:0,他引:3
The reconstitution of functional sarcoplasmic reticulum vesicles capable of Ca2+ transport has been achieved. Sarcoplasmic reticulum vesicles are first solubilized with deoxycholate and then reassembled into membranous vesicles by removal of the detergent using dialysis. The Ca2+ pump protein can, by itself, be reconstituted to form membranous vesicles capable of energized Ca2+ binding and uptake. The lipid content of the reconstituted vesicles is about the same as that of the original sarcoplasmic reticulum vesicles. The reconstituted vesicles have an elevated ATPase activity. Ca2+ binding and uptake in the presence of ATP are restored to about 25% and 50%, respectively. 相似文献
7.
J W Schmidt T R Hinds F F Vincenzi 《Comparative biochemistry and physiology. A, Comparative physiology》1985,82(3):601-607
Ca2+-pump ATPase activities of membranes isolated from human and dog RBCs were compared under a variety of conditions. Specific activity of the dog enzyme was less than that of human. Unlike the human enzyme, the dog Ca2+-pump ATPase was not stimulated by exogenously added calmodulin (CaM) or oleate. The Ca2+ dependence of the dog Ca2+-pump ATPase resembled that of the CaM-activated form of the human enzyme. Cross-linking of Azido-125I-CaM to dog RBC membranes did not label a Ca2+-pump ATPase of molecular weight similar to that found in human RBC membranes. It is suggested that the Ca2+-pump ATPase in isolated dog RBC membranes exists in an activated state, not due to endogenous CaM, but possibly due to partial proteolysis. 相似文献
8.
Horacio Guimarães-Motta Leopoldo De Meis 《Archives of biochemistry and biophysics》1980,203(1):395-403
Millisecond mixing and quenching experiments were performed in order to study the rate of phosphorylation by Pi of the Ca2+-dependent ATPase of sarcoplasmic reticulum vesicles. A rapid phosphoenzyme formation was observed when the vesicles were preincubated in the absence of Ca2+ prior to the addition of Pi and Mg2+ to the medium, the half-time being in the range of 6 to 10 ms. A lag phase and a 5- to 10-fold slower rate of phosphoenzyme formation were observed when the enzyme was preincubated with Ca2+ prior to the addition to the reaction mixture of Pi, Mg2+, and an excess of ethylene glycol bis(β-aminoethyl ether)N,N′-tetraacetic acid. The rate of phosphoenzyme hydrolysis was measured either by the addition of Ca2+ or, in the absence of Ca2+, by tracing the hydrolysis of radioactive phosphoenzyme upon the addition of nonradioactive Pi. In the presence of Ca2+, the rate of phosphoenzyme hydrolysis was found to be one order of magnitude slower than the rate of hydrolysis measured in the absence of Ca2+. Different rates of phosphoenzyme formation and cleavage were found depending on whether sarcoplasmic reticulum vesicles or purified Ca2+-dependent ATPase were used. A transient phosphorylation by Pi was observed when the enzyme was preincubated in the absence of Ca2+ and then added to a medium containing Pi, Mg2+, and excess of Ca2+. The enzyme was phosphorylated during the initial 100 ms, the phosphoenzyme formed being slowly hydrolyzed in the subsequent incubation intervals. In these conditions ATP synthesis was observed if ADP was added to the mixture 100 ms after starting the reaction. No transient phosphorylation by Pi was observed when the enzyme was preincubated with Ca2+. Synthesis of a small but significant amount of ATP was observed when the enzyme was preincubated in the absence of Ca2+ and then added to a medium containing Pi, ADP, Mg2+, and 20 mm CaCl2. This was not observed when the enzyme was preincubated in the presence of Ca2+. 相似文献
9.
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
10.
Kannan Subburaj Elimban Vijayan Fandrich Robert R. Kardami Elissavet Dhalla Naranjan S. 《Molecular and cellular biochemistry》1999,197(1-2):187-194
Cardiac plasma membrane Ca2+/Mg2+ ecto-ATPase (myoglein) requires millimolar concentrations of either Ca2+ or Mg2+ for maximal activity. In this paper, we report its localization by employing an antiserum raised against the purified rat cardiac Ca2+/Mg2+ ATPase. As assessed by Western blot analysis, the antiserum and the purified immunoglobulin were specific for Ca2+/Mg2+ ecto-ATPase; no cross reaction was observed towards other membrane bound enzymes such as cardiac sarcoplasmic reticulum Ca2+-pump ATPase or sarcolemmal Ca2+-pump ATPase. On the other hand, the cardiac Ca2+/Mg2+ ecto-ATPase was not recognized by antibodies specific for either cardiac sarcoplasmic reticulum Ca2+-pump ATPase or plasma membrane Ca2+-pump ATPase. Furthermore, the immune serum inhibited the Ca2+/Mg2+ ecto-ATPase activity of the purified enzyme preparation. Immunofluorescence of cardiac tissue sections and neonatal cultured cardiomyocytes with the Ca2+/Mg2+ ecto-ATPase antibodies indicated the localization of Ca2+/Mg2+ ecto-ATPase in association with the plasma membrane of myocytes, in areas of cell-matrix or cell-cell contact. Staining for the Ca2+/Mg2+ ecto-ATPase was not cardiac specific since the antibodies detected the presence of membrane proteins in sections from skeletal muscle, brain, liver and kidney. The results indicate that Ca2+/Mg2+ ecto-ATPase is localized to the plasma membranes of cardiomyocytes as well as other tissues such as brain, liver, kidney and skeletal muscle. 相似文献
11.
Functional Reconstitution of an ATP-Driven Ca-Transport System from the Plasma Membrane of Commelina communis L
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The protein(s) that constitute(s) the ATP-driven Ca2+-translocator of plasma membrane enriched vesicles obtained by aqueous two-phase partitioning from leaves of Commelina communis L. has/have been solubilized and reincorporated into tightly sealed liposomes. The reconstituted Ca2+-transport system was studied using ATP-driven 45Ca2+ import into the proteoliposomes as a measure of activity. The detergent, 3-[(3-cholamidopropyl) dimethylammonio]-1-propane-sulfonate proved to be the most suitable and was used at 10 millimolar concentration, i.e. just above its critical micellar concentration. The presence of additional phospholipid (2 milligrams phosphatidylcholine per milliliter) and ATP (5 millimolar) improved the solubilization and/or reconstitution. The characteristics of the reconstituted system were similar to those of the plasma membrane-bound activity, including the apparent Km for Ca2+ (5.2 micromolar), inhibition by relatively high levels of vanadate (IC50 = 500 micromolar) and lacking response to added calmodulin. The reconstituted transport system was very strongly inhibited by erythrosine B (IC50 = 0.01 micromolar) and had a low apparent Km for ATP (11.4 micromolar). As in the plasma membrane vesicles, the protonophore carbonylcyanide m-chlorophenyl hydrazone did not affect Ca2+-transport detectably in the reconstituted system. However, low levels of the Ca2+-ionophore A 23187 instantaneously discharged 90% of the Ca2+ associated with the vesicles, proving that it had been accumulated in the intravesicular volume in soluble, freely exchangeable form. Ca2+-transport in the reconstituted system was thus primary active, through a Ca2+-translocating ATPase. The system reported here may serve as a valuable tool for purifying the Ca2+-ATPase and for studying structural and functional aspects of the purified enzyme. 相似文献
12.
The effects of anti-calmodulin (CaM) serum, the CaM antagonist W7-agarose, the Ca2+ chelator ethyleneglycol-bis-(β-aminoethyl)-N,N,N′,N′-tetraacetic acid (EGTA) and exogenous pure CaM on pollen germination
and tube growth of Hippeastrum rutilum Herb were studied. Pollen germination and tube growth were inhibited or completely stopped by anti-CaM serum in a dose-dependent
manner, while the same amount of preimmune serum had no effect on either process. Pollen germination and tube growth were
also inhibited or completely stopped by the CaM antagonist W7-agarose and the Ca2+ chelator EGTA. The addition of exogenous pure CaM enhanced pollen germination and tube growth, whereas the same amount of
bovine serum albumin had no effect. The inhibitory effects caused by anti-CaM serum, W7-agarose and EGTA-washing could be
reversed completely by the addition of exogenous pure CaM. These results indicate that extracellular CaM initiates pollen
germination and tube growth, whereas exogenous CaM enhances the above processes, and may provide a novel view for understanding
the control of pollen germination and tube growth.
Received: 12 December 1996 / Accepted: 15 January 1997 相似文献
13.
We have previously proposed a role for calmodulin (CaM) in the regulation of initiation of Ca2+ entry in Jurkat T cells, as well as in the regulation of the current that mediates Ca2+ entry, IT. In this report, we provide evidence for the mechanism of CaM action. We have previously shown that activation-induced Ca 2+ entry into Jurkat T cells is mediated by a current we have called IT. In the whole cell variation, but not the perforated patch variation, of the patch clamp technique, this current is short-lived (under 6 min) suggesting that the current is under the control of a diffusible component of the cytosol. Addition of CaM to the whole cell recording pipette solution maintained IT for up to 20 min, suggesting that CaM may be this diffusible component. Pharmacological inhibitors of CaM blocked the augmentation of IT normally induced by an activating stimulus. Cells electroporated in the presence of anti-CaM antibodies had reduced influx of extracellular Ca2+, with no change in release of Ca2+ from the internal stores. These observations suggest that T cell receptor engagement initiates Ca2+ influx by a pathway that likely includes CaM, which may in turn regulate IT. Influx of extracellular Ca2+ is required for cellular proliferation, and inhibition of CaM by pharmacological inhibitors reduced cellular proliferation. This same inhibition of proliferation was seen in cells electroporated with anti-CaM antibodies. This suggests that inhibition of CaM and/or IT may be a target for therapeutic inhibition of inappropriate T cell proliferation. 相似文献
14.
Gethyn J. Allen Francisco Morais Shelagh Muir R. Gareth Wyn Jones Roger A. Leigh 《Physiologia plantarum》1995,94(3):506-514
Microsomal fractions from wheat tissues exhibit a higher level of ATP hydrolytic activity in the presence of Ca2+ than Mg2+. Here we characterise the Ca2+-dependent activity from roots of Triticum aestivum lev. Troy) and investigate its possible function. Ca2+-dependent ATP hydrolysis in the microsomal fraction occurs over a wide pH range with two slight optima at pH 5.5 and 7.5. At these pHs the activity co-migrates with the major peak of nitrate-inhibited Mg2+. Cl-ATPase on continuous sucrose gradients indicating that it is associated with the vacuolar membrane. Ca2+-dependent ATP hydrolysis can be distinguished from an inhibitory effect of Ca2+ on the plasma membrane K+, Mg2+-ATPase following microsomal membrane separation using aqueous polymer two phase partitioning. The Ca2+-dependent activity is stimulated by free Ca2+ with a Km of 8.1 μM in the absence of Mg2+ ([CaATP] = 0.8 mM). Vacuoiar membrane vacuolar preparations contain a higher Ca2+-dependent than Mg2+-dependent ATP hydrolysis, although the two activities are not directly additive. The nucleotide specificity of the divalent ion-dependent activities in vacuolar membrane-enriched fractions was low. hydrolysis of CTP and UTP being greater than ATP hydrolysis with both Ca2+ and Mg2+ The Ca2+-dependent activity did discriminate against dinucleotides, and mononucleotides. and failed to hydrolyse phosphatase substrates. Despite low nucleotide specificity the Mg2+-dependent activity functioned as a bafilomycin sensitive H+-pump in vacuolar membrane vesicles. Ca2+-dependent ATP hydrolysis was not inhibited by the V-, P-, or F-type ATPase inhibitors bafilomycin. vanadate and azide, respectively. nor by the phosphatase inhibitor molybdate, but was inhibited 20% at pH 7.5 by K+. Possible functions of Ca2+-dependent hydrolysis as a H+-pump or a Ca2+-pump was investigated using vacuolar membrane vesicles. No H+ or Ca2+ translocating activity was observed under conditions when the Ca2+-dependent ATP hydrolysis was active. 相似文献
15.
A procedure for the isolation of highly purified sarcoplasmic reticulum vesicles from rabbit skeletal muscle has been described using sucrose gradient centrifugation in zonal rotors. The yield of our purest fraction was 300 mg of sarcoplasmic reticulum protein using 1 kg muscle. The sarcoplasmic reticulum vesicles were relatively simple in composition. The Ca2+-pump protein accounted for most (approx. two-thirds) of the sarcoplasmic reticulum protein. Two other protein components, a Ca2+-binding protein and a M55 protein (approx. 55 000 daltons) each accounted for about 5–10% of the protein. Enrichment in the level of phosphoenzyme by the Ca2+-pump protein was regarded as an important index of the purification of sarcoplasmic reticulum vesicles. The sarcoplasmic reticulum vesicles were capable of forming 6.4 nmoles of 32P-labelled phosphoenzyme per mg protein and had a high capacity of energized Ca2+ uptake. The Ca2+-dependent formation of phosphoenzyme has been used to estimate the sarcoplasmic reticulum protein content in rabbit skeletal muscle and found to be about 2.5% of the total muscle protein.The Ca2+-pump and Ca2+-binding proteins were isolated with a purity of 90% or more by treating the purified sarcoplasmic reticulum vesicles with bile acids in the presence of salt. The solubilized Ca2+-pump protein reaggregated during dialysis together with phospholipid to form membranous vesicles which were capable of forming approx. 9 nmoles 32P-labelled phosphoenzyme per mg protein. The Ca2+-binding protein was water soluble and contained a high percentage of acidic amino acids (35% of total residues).Ca2+ binding by sarcoplasmic reticulum vesicles and by the Ca2+-pump and Ca2+-binding proteins was studied by equilibrium dialysis. Sarcoplasmic reticulum vesicles and Ca2+-pump protein contained nonspecific high-affinity Ca2+ binding sites with a capacity of 90–100 and 55–70 nmoles Ca2+ per mg protein, respectively. Both of them specifically bound 10–15 nmoles Ca2+ per mg protein. The binding constants for nonspecific and specific Ca2+ binding by both preparations were approx. 1 μM?1. The Ca2+-binding protein nonspecifically bound 900–1000 nmoles Ca2+ per mg protein with a binding constant of about 0.25 μM?1. 相似文献
16.
Xueting Jiang Yongbin Gao Hongsheng Zhou Jianqing Chen Juyou Wu Shaoling Zhang 《Plant cell reports》2014,33(2):255-263
Key message
This study indicated that Ca 2+ , ROS and actin filaments were involved with CaM in regulating pollen tube growth and providing a potential way for overcoming pear self-incompatibility.Abstract
Calmodulin (CaM) has been associated with various physiological and developmental processes in plants, including pollen tube growth. In this study, we showed that CaM regulated the pear pollen tube growth in a concentration-dependent bi-phasic response. Using a whole-cell patch-clamp configuration, we showed that apoplastic CaM induced a hyperpolarization-activated calcium ion (Ca2+) current, and anti-CaM largely inhibited this type of Ca2+ current. Moreover, upon anti-CaM treatment, the reactive oxygen species (ROS) concentration decreased and actin filaments depolymerized in the pollen tube. Interestingly, CaM could partially rescue the inhibition of self-incompatible pear pollen tube growth. This phenotype could be mediated by CaM-enhanced pollen plasma membrane Ca2+ current, tip-localized ROS concentration and stabilized actin filaments. These data indicated that Ca2+, ROS and actin filaments were involved with CaM in regulating pollen tube growth and provide a potential way for overcoming pear self-incompatibility. 相似文献17.
The effects of calmodulin (CaM) on ATPase activity and ATP-dependent formation of a proton gradient (ΔpH) were studied in tonoplast membrane vesicles from corn (Zea mays L.) roots. At 0.6 micromolar, CaM stimulated ATPase activity by about 20% in the absence of an uncoupler, but by only 4% in its presence. Thus, the uncoupler-dependent increment of activity was decreased 30 to 45% by CaM. The formation of a proton gradient across the membrane vesicle, measured by quinacrine fluorescence quench, was inhibited about 20% by CaM. Its effect was additive to the effect of Ca2+ and was completely abolished by EGTA. These effects of CaM could be due to stimulation of H+ efflux or due to inhibition of the H+-ATPase. To distinguish between these possibilities, we examined the effect of CaM on dissipation of preformed ΔpH after the ATPase was inhibited. CaM stimulated the dissipation of a preformed ΔpH by 40% after the H+-ATPase was inhibited with NO3−. This indicates that CaM facilitates the recycling of protons across the tonoplast membranes and does not regulate the H+-ATPase by direct inhibition. 相似文献
18.
Sphingosylphosphorylcholine (SPC), a lipid mediator with putative second messenger functions, has been reported to regulate ryanodine receptors (RyRs), Ca2+ channels of the sarco/endoplasmic reticulum. RyRs are also regulated by the ubiquitous Ca2+ sensor calmodulin (CaM), and we have previously shown that SPC disrupts the complex of CaM and the peptide corresponding to the CaM-binding domain of the skeletal muscle Ca2+ release channel (RyR1). Here we report that SPC also displaces Ca2+-bound CaM from the intact RyR1, which we hypothesized might lead to channel activation by relieving the negative feedback Ca2+CaM exerts on the channel. We could not demonstrate such channel activation as we have found that SPC has a direct, CaM-independent inhibitory effect on channel activity, confirmed by both single channel measurements and [3H]ryanodine binding assays. In the presence of Ca2+CaM, however, the addition of SPC did not reduce [3H]ryanodine binding, which we could explain by assuming that the direct inhibitory action of the sphingolipid was negated by the simultaneous displacement of inhibitory Ca2+CaM. Additional experiments revealed that RyRs are unlikely to be responsible for SPC-elicited Ca2+ release from brain microsomes, and that SPC does not exert detergent-like effects on sarcoplasmic reticulum vesicles. We conclude that regulation of RyRs by SPC involves both CaM-dependent and -independent mechanisms, thus, the sphingolipid might play a physiological role in RyR regulation, but channel activation previously attributed to SPC is unlikely. 相似文献
19.
Irene Mangialavori Mariela Ferreira-Gomes Mar��a F. Pignataro Emanuel E. Strehler Juan Pablo F. C. Rossi 《The Journal of biological chemistry》2010,285(1):123-130
The purpose of this work was to obtain information about conformational changes of the plasma membrane Ca2+-pump (PMCA) in the membrane region upon interaction with Ca2+, calmodulin (CaM) and acidic phospholipids. To this end, we have quantified labeling of PMCA with the photoactivatable phosphatidylcholine analog [125I]TID-PC/16, measuring the shift of conformation E2 to the auto-inhibited conformation E1I and to the activated E1A state, titrating the effect of Ca2+ under different conditions. Using a similar approach, we also determined the CaM-PMCA dissociation constant. The results indicate that the PMCA possesses a high affinity site for Ca2+ regardless of the presence or absence of activators. Modulation of pump activity is exerted through the C-terminal domain, which induces an apparent auto-inhibited conformation for Ca2+ transport but does not modify the affinity for Ca2+ at the transmembrane domain. The C-terminal domain is affected by CaM and CaM-like treatments driving the auto-inhibited conformation E1I to the activated E1A conformation and thus modulating the transport of Ca2+. This is reflected in the different apparent constants for Ca2+ in the absence of CaM (calculated by Ca2+-ATPase activity) that sharply contrast with the lack of variation of the affinity for the Ca2+ site at equilibrium. This is the first time that equilibrium constants for the dissociation of Ca2+ and CaM ligands from PMCA complexes are measured through the change of transmembrane conformations of the pump. The data further suggest that the transmembrane domain of the PMCA undergoes major rearrangements resulting in altered lipid accessibility upon Ca2+ binding and activation. 相似文献
20.
Stimulation of the calmodulin-independent activity of the red cell Ca2+-pump ATPase by trypsin treatment (of calmodulin free red cell membranes) is sensitive to Ca2+ in a concentration range near the KCa of the transport site. The Ca2+ requirement for this effect is absolute, whereas the calmodulin sensitivity of the ATPase can be abolished by sufficient trypsin attack in the absence of Ca2+, although Ca2+ accelerates inactivation. This indicates that the two effects of trypsin are due to at least two distinct cleavage sites in the pump protein. 相似文献