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1.
We describe the design, characterization and application of a new genetically encoded fluorescent biosensor for intracellular detection of both free Ca(2+)-calmodulin and apocalmodulin, which together comprise the available calmodulin concentration. The biosensor binds both forms of calmodulin with an apparent Kd value of 3 microM, and has kinetic properties making it suitable for monitoring dynamic changes on a subsecond time scale. It can be used in conjunction with the fluorescent Ca(2+)-indicator, indo-1, allowing the available calmodulin and free Ca2+ concentrations to be monitored concurrently. We have determined an intracellular available calmodulin concentration of 8.8 +/- 2.2 microM under resting conditions in a human kidney cell line stably expressing the biosensor. Elevation of the intracellular free Ca2+ concentration by agonist, store-operated Ca(2+)-entry or ionophore results in Ca(2+)-dependent consumption of the available calmodulin. A plot of normalized values for the available calmodulin concentration versus the free Ca2+ concentration fits a consumption curve with a cooperativity coefficient of 1.8 and a [Ca2+]50 of 850 nM. There is no detectible binding of calmodulin to the biosensor above a free Ca2+ concentration of approximately 4 microM, consistent with an available calmodulin concentration < or = 200 nM under these conditions, and an overall excess of calmodulin-binding sites.  相似文献   

2.
The Ca2+-ATPase (ATP phosphohydrolase, EC 3.6.1.3) in human erythrocyte membranes, which is part of the Ca2+ pump, can be activated by binding of calmodulin. Rate constants (k1) for association of calmodulin and enzyme, which depends on the Ca2+ concentration, have been determined by the aid of an enzyme model. k1 increased from 0.25 . 10(6) to 17.3 . 10(6) M-1 . min-1 (70 times) when the free Ca2+ concentration was raised from 0.7 to 20 microM. The binding of calmodulin to the Ca2+-ATPase is reversible. The rate constants (k-1) for dissociation of enzyme-calmodulin complex decreased from 6.0 to 0.044 min-1 (135 times) when the free Ca2+ concentration was increased from 0.1 to 2-20 microM. The apparent dissociation constant Kd = k-1/k1 accordingly increased from 2.5 nM to 25 microM (or higher) when the Ca2+ concentration was reduced from 20 to 0.1 microM. Therefore, at 10(-7) M free Ca2+ most of the Ca2+-pump enzyme will not bind calmodulin. For the intact cell the time dependences of activation and deactivation of the Ca2+-pump enzyme have been estimated from the rate constants above. The results suggest that the Ca2+ pump is well suited to maintain a cytosolic concentration of 10(-7) M free Ca2+ (or lower) in the unstimulated cell and, when the cell is stimulated, to allow transient Ca2+ signals up to approx. 10(-5) M in the cytosol.  相似文献   

3.
Black DJ  Leonard J  Persechini A 《Biochemistry》2006,45(22):6987-6995
The relationship between the free Ca2+ concentration and the apparent dissociation constant for the complex between calmodulin (CaM) and the neuromodulin IQ domain consists of two phases. In the first phase, Ca2+ bound to the C-ter EF hand pair in CaM increases the Kd for the complex from the Ca2+-free value of 2.3 +/- 0.1 microM to a value of 14.4 +/- 1.3 microM. In the second phase, Ca2+ bound to the N-ter EF hand pair reduces the Kd for the complex to a value of 2.5 +/- 0.1 microM, reversing the effect of the first phase. Due to energy coupling effects associated with these phases, the mean dissociation constant for binding of Ca2+ to the C-ter EF hand pair is increased approximately 3-fold, from 1.8 +/- 0.1 to 5.1 +/- 0.7 microM, and the mean dissociation constant for binding of Ca2+ to the N-ter EF hand pair is decreased by the same factor, from 11.2 +/- 1.0 to 3.5 +/- 0.6 microM. These characteristics produce a bell-shaped relationship between the apparent dissociation constant for the complex and the free Ca2+ concentration, with a distance of 5-6 microM between the midpoints of the rising and falling phases. Release of CaM from the neuromodulin IQ domain therefore appears to be promoted over a relatively narrow range of free Ca2+ concentrations. Our results demonstrate that CaM-IQ domain complexes can function as biphasic Ca2+ switches through opposing effects of Ca2+ bound sequentially to the two EF hand pairs in CaM.  相似文献   

4.
Measurements of cellular Ca2+-calmodulin concentrations have suggested that competition for limiting calmodulin may couple calmodulin-dependent activities. Here we have directly tested this hypothesis. We have found that in endothelial cells the amount of calmodulin bound to nitric-oxide synthase and the catalytic activity of the enzyme both are increased approximately 3-fold upon changes in the phosphorylation status of the enzyme. Quantitative immunoblotting indicates that the synthase can bind up to 25% of the total cellular calmodulin. Consistent with this, simultaneous determinations of the free Ca2+ and Ca2+-calmodulin concentrations in these cells performed using indo-1 and a fluorescent calmodulin biosensor (Kd = 2 nm) indicate that increased binding of calmodulin to the synthase is associated with substantial reductions in the Ca2+-calmodulin concentrations produced and an increase in the [Ca2+]50 for formation of the calmodulin-biosensor complex. The physiological significance of these effects is confirmed by a corresponding 40% reduction in calmodulin-dependent plasma membrane Ca2+ pump activity. An identical reduction in pump activity is produced by expression of a high affinity (Kd = 0.3 nm) calmodulin biosensor, and treatment to increase calmodulin binding to the synthase then has no further effect. This suggests that the observed reduction in pump activity is due specifically to reduced calmodulin availability. Increases in synthase activity thus appear to be coupled to decreases in the activities of other calmodulin targets through reductions in the size of a limiting pool of available calmodulin. This exemplifies what is likely to be a ubiquitous mechanism for coupling among diverse calmodulin-dependent activities.  相似文献   

5.
A high degree of ATP hydrolytic activity present in purified rat pancreatic acinar cells was localized to plasma membranes. This activity was stimulated almost equally by Mg2+ or Ca2+. Kinetic analysis revealed that the enzyme had a higher affinity for Ca2+ (Kd = 1.73 microM) than Mg2+ (Kd = 2.98 microM) but a similar maximal rate of activity. A comparison of substrate requirements revealed very similar profiles for the Mg2+- and Ca2+-stimulated activities. Combinations of saturating concentrations of Mg2+ or Ca2+ produced the same degree of maximal activity. Investigation of the partial reactions of the ATPase activity revealed two phosphoprotein intermediates (Mr = 115,000 and 130,000) in the presence of Ca2+ and Mg2+. A significant stimulation of the Ca2+-ATPase activity by calmodulin was observed (Kd = 0.7 microM). Calmodulin increased the Ca2+-sensitivity of this enzyme system; Mg2+ appeared to be required for this effect. The Ca2+-ATPase activity was also stimulated by acidic phospholipids. Using an 125I-labeled calmodulin gel overlay technique, calmodulin was shown to bind in a Ca2+-dependent fashion to 133,000- and 230,000-dalton proteins present in the plasma membrane-enriched fraction. Under conditions that favor Ca2+-dependent kinase activity, calmodulin enhanced the phosphorylation of a 30,000- and 19,000-dalton protein. The major ATP hydrolytic activity in pancreatic acinar plasma membranes was present as an ectoenzyme.  相似文献   

6.
Low concentrations of free Ca2+ stimulated the hydrolysis of ATP by plasma membrane vesicles purified from guinea pig neutrophils and incubated in 100 mM HEPES/triethanolamine, pH 7.25. In the absence of exogenous magnesium, apparent values obtained were 320 nM (EC50 for free Ca2+), 17.7 nmol of Pi/mg X min (Vmax), and 26 microM (Km for total ATP). Studies using trans- 1,2-diaminocyclohexane- N,N,N',N',-tetraacetic acid as a chelator showed this activity was dependent on 13 microM magnesium, endogenous to the medium plus membranes. Without added Mg2+, Ca2+ stimulated the hydrolysis of several other nucleotides: ATP congruent to GTP congruent to CTP congruent to ITP greater than UTP, but Ca2+-stimulated ATPase was not coupled to uptake of Ca2+, even in the presence of 5 mM oxalate. When 1 mM MgCl2 was added, the vesicles demonstrated oxalate and ATP-dependent calcium uptake at approximately 8 nmol of Ca2+/mg X min (based on total membrane protein). Ca2+ uptake increased to a maximum of approximately 17-20 nmol of Ca2+/mg X min when KCl replaced HEPES/triethanolamine in the buffer. In the presence of both KCl and MgCl2, Ca2+ stimulated the hydrolysis of ATP selectively over other nucleotides. Apparent values obtained for the Ca2+-stimulated ATPase were 440 nM (EC50 for free Ca2+), 17.5 nmol Pi/mg X min (Vmax) and 100 microM (Km for total ATP). Similar values were found for Ca2+ uptake which was coupled efficiently to Ca2+-stimulated ATPase with a molar ratio of 2.1 +/- 0.1. Exogenous calmodulin had no effect on the Vmax or EC50 for free Ca2+ of the Ca2+-stimulated ATPase, either in the presence or absence of added Mg2+, with or without an ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N',-tetraacetic acid pretreatment of the vesicles. The data demonstrate that calcium stimulates ATP hydrolysis by neutrophil plasma membranes that is coupled optimally to transport of Ca2+ in the presence of concentrations of K+ and Mg2+ that appear to mimic intracellular levels.  相似文献   

7.
Measurement of the volume change by a rapid density method upon sequential addition of calcium ion to calmodulin showed relatively large, nonuniform increases for the first 4 moles Ca2+ per mole calmodulin. Substantially larger volume increases (approximately 15 ml/mol protein) were observed upon addition of the second and fourth moles Ca2+ relative to the first and third moles added per mole calmodulin. A total volume increase of approximately 170 ml/mol protein attended the addition of 4 moles Ca2+, as expected for multidentate carboxylate coordination to metal ion. Marginal changes in volume were observed upon further additions, the data showing a remarkably sharp transition after [Ca2+]/[calmodulin] = 4. The results are consistent with an ordered binding of Ca2+ in which pair-wise additions produce similar volume changes; the volume change behavior, however, does not indicate an absence of distinct conformational states for a Ca2+(1)-calmodulin and a Ca2+(3)-calmodulin complex as has been proposed on the basis of 1H-NMR evidences.  相似文献   

8.
The calmodulin activation of the (Ca2+ + Mg2+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) in human erythrocyte membranes was studied in the range of 1 nM to 40 microM of purified calmodulin. The apparent calmodulin-affinity of the ATPase was strongly dependent on Ca2+ and decreased approx. 1000-times when the Ca2+ concentration was reduced from 112 to 0.5 microM. The data of calmodulin (Z) activation were analyzed by the aid of a kinetic enzyme model which suggests that 1 molecule of calmodulin binds per ATPase unit and that the affinities of the calcium-calmodulin complexes (CaiZ) decreases in the order of Ca3Z greater than Ca4Z greater than Ca2Z greater than or equal to CaZ. Furthermore, calmodulin dissociates from the calmodulin-saturated Ca2+-ATPase in the range of 10(-7)-10(-6) M Ca2+, even at a calmodulin concentration of 5 microM. The apparent concentration of calmodulin in the erythrocyte cytosol was determined to be 3 to 5 microM, corresponding to 50-80-times the cellular concentration of Ca2+-ATPase, estimated to be approx. 10 nmol/h membrane protein. We therefore conclude that most of the calmodulin is dissociated from the Ca2+-transport ATPase in erythrocytes at the prevailing Ca2+ concentration (probably 10(-7)-10(-8) M) in vivo, and that the calmodulin-binding and subsequent activation of the Ca2+-ATPase requires that the Ca2+ concentration rises to 10(-6)-10(-5) M.  相似文献   

9.
Plant and fungal calmodulin: Ca2+-dependent regulation of plant NAD kinase   总被引:3,自引:0,他引:3  
Although little is known about the role(s) of second messengers, including free Ca2+, in plant cells there has been increasing evidence for a role for Ca2+ in metabolic regulation in plants. The recent demonstration that the Ca2+-binding protein, calmodulin exists in extracts of higher plants and basidiomycete fungi provides a basis for understanding Ca2+-dependent metabolic regulation in plant cells. In this review we summarize the similarities and differences of plant, fungal and mammalian calmodulin. We also discuss the known in vitro functions of calmodulin in higher plants. A Ca2+-calmodulin-dependent NAD kinase has been purified to homogeneity from extracts of pea seedlings and shown to be absolutely dependent upon calmodulin and microM levels of free Ca2+ for activity. The available evidence suggest that this Ca2+-calmodulin-dependent NAD kinase is the major form of plant NAD kinase and that this regulatory enzyme is localized in the chloroplast. A model is presented which predicts that the rate of photosynthesis is regulated by a receptor-mediated change in the level of chloroplastic free Ca2+ upon illumination. Free Ca2+, acting as a second messenger, forms a Ca2+-calmodulin complex thus converting calmodulin to its active conformation. This Ca2+-calmodulin complex then activates chloroplastic NAD kinase resulting in an increased NADP/NAD ratio.  相似文献   

10.
The fluorescent Ca2+ indicator, quin 2, has been used in isolated striated muscle fibres. There is a distinct quin 2 fluorescence peak at lambda 500 nm upon excitation at lambda 339 nm after axial injection of the potassium salt of quin 2, pH 7.1. Single voltage-clamp or current clamp electrical stimulation resulted in a distinct transient change in the fluorescence at lambda 500 nm which was not observed at lambda 400 nm, the peak of the fibre autofluorescence. Ca2+ buffering is marked at high quin 2 concentrations (greater than or equal to 400 microM) producing a slow decay of force and fluorescence. At lower concentrations (8-30 microM) of quin, the decay of force is within the range observed in non-injected control fibres. A Kd of 457 nM at 5 mM free Mg2+ suggests an upper resting free Ca2+ concentration of 310 nM at 12 degrees C.  相似文献   

11.
Incubation of bovine aortic native actomyosin with cyclic AMP and bovine aortic cyclic AMP-dependent protein kinase produced a rightward shift in the relation between free Ca2+ and both superprecipitation and actomyosin ATPase activity. The relation between free Ca2+ and phosphorylation of myosin light chains was also shifted to the right. The concentration of free Ca2+ required for half-maximal activation of both ATPase activity and myosin light chain phosphorylation was approximately 1.0 microM for control actomyosin and 2.5 microM for actomyosin incubated with cyclic AMP-protein kinase. Neither basal nor maximal activities were significantly affected by incubation with cyclic AMP-protein kinase. Addition of e microM calmodulin to cyclic AMP-protein kinase-treated actomyosin relieved inhibition of both superprecipitation and myosin light chain phosphorylation. These findings suggest that cyclic AMP-protein kinase-mediated inhibition of actin-myosin interactions in vascular smooth muscle involve a shift in the Ca2+ sensitivity of the system. This shift probably involves Ca2+-calmodulin interactions and the control of phosphorylation of the myosin light chains.  相似文献   

12.
To identify protein targets for calmodulin (CaM) in the cilia of Paramecium tetraurelia, we employed a 125I-CaM blot assay after resolution of ciliary proteins on SDS/polyacrylamide gels. Two distinct types of CaM-binding proteins were detected. One group bound 125I-CaM at free Ca2+ concentrations above 0.5-1 microM and included a major binding activity of 63 kDa (C63) and activities of 126 kDa (C126), 96 kDa (C96), and 36 kDa (C36). CaM bound these proteins with high (nanomolar) affinity and specificity relative to related Ca2+ receptors. The second type of protein bound 125I-CaM only when the free Ca2+ concentration was below 1-2 microM and included polypeptides of 95 kDa (E95) and 105 kDa (E105). E105 may also contain Ca2+-dependent binding sites for CaM. Both E95 and E105 exhibited strong specificity for Paramecium CaM over bovine CaM. Ciliary subfractionation experiments suggested that C63, C126, C96, E95, and E105 are bound to the axoneme, whereas C36 is a soluble and/or membrane-associated protein. Additional Ca2+-dependent CaM-binding proteins of 63, 70, and 120 kDa were found associated with ciliary membrane vesicles. In support of these results, filtration binding assays also indicated high-affinity binding sites for CaM on isolated intact axonemes and suggested the presence of both Ca2+-dependent and Ca2+-inhibitable targets. Like E95 and E105, the Ca2+-inhibitable CaM-binding sites showed strong preference for Paramecium CaM over vertebrate CaM and troponin C. Together, these results suggest that CaM has multiple targets in the cilium and hence may regulate ciliary motility in a complex and pleiotropic fashion.  相似文献   

13.
The binding parameters of 125I-labeled calmodulin to bovine cerebellar membranes have been determined and correlated with the activation of adenylate cyclase by calmodulin. In the presence of saturating levels of free Ca2+ calmodulin binds to a finite number of specific membrane sites with a dissociation constant (Kd) of 1.2 nM. Furthermore, Scatchard analysis reveals a second population of binding sites with a 100-fold lower affinity for calmodulin. The Ca2+-dependence of calmodulin binding and of adenylate cyclase activation varies with the amount of calmodulin present, as can be inferred from the model of sequential equilibrium reactions which describes the activation of calmodulin-dependent enzymes. On the basis of this model, a quantitative analysis of the effect of free Ca2+ and of free calmodulin concentration on both binding and activation of adenylate cyclase was carried out. This analysis shows that both processes take place only when calmodulin is complexed with at least three Ca2+ atoms. The concentration of the active calmodulin X Ca2+ species required for half-maximal activation of adenylate cyclase is very similar to the Kd of the high affinity binding sites on brain membranes. A Hill coefficient of approx. 1 was found for both processes indicating an absence of cooperativity. Phenothiazines and thioxanthenes antipsychotic agents inhibit calmodulin binding to membranes and calmodulin-dependent activation of adenylate cyclase with a similar order of potency. These results suggest that the Ca2+-dependent binding of calmodulin to specific high affinity sites on brain membranes regulates the activation of adenylate cyclase by calmodulin.  相似文献   

14.
Inside-out vesicles of human erythrocytes took up Ca2+ against an electrochemical gradient. This Ca2+ uptake was dependent on ATP and was stimulated by calmodulin. Treatment of vesicles with 1 mM-EDTA exposed an apparent low-CA2+-affinity Ca2+-transport component with Kd of about 100 microM-Ca2+ or more. This was converted into a single high-Ca2+-affinity transport activity of Kd about 2.5 microM-Ca2+ in the presence of 2 micrograms of calmodulin/ml, showing that the decrease in transport activity after EDTA treatment was reversible. Vesicles not extracted with EDTA showed mainly apparent high-Ca2+-affinity kinetics even in the absence of added calmodulin. Trifluoperazine (30 microM) and calmodulin-binding protein (20 micrograms/ml) inhibited about 50% of the high-affinity Ca2+ uptake and (Ca2+ + Mg2+)-ATPase (Ca2+-activated, Mg2+-dependent ATPase) activity of these vesicles, indicating that the vesicles isolated by the procedure used retained some calmodulin from the erythrocytes. Comparison of Ca2+ transport and (Ca2+ + Mg2+)-ATPase activities in inside-out vesicles yielded a variable Ca2+/P1 stoichiometric ratio. At low free Ca2+ concentrations (below 20 micro-Ca2+), a Ca2+/P1 ration of about 2 was found, whereas at higher Ca2+ concentrations the stoichiometry was approx. 1. The stoichiometry was not significantly altered by calmodulin.  相似文献   

15.
Calmodulin inhibits inositol 1,4,5-trisphosphate (IP3) binding to the IP3 receptor in both a Ca2+-dependent and a Ca2+-independent way. Because there are no functional data on the modulation of the IP3-induced Ca2+ release by calmodulin at various Ca2+ concentrations, we have studied how cytosolic Ca2+ and Sr2+ interfere with the effects of calmodulin on the IP3-induced Ca2+ release in permeabilized A7r5 cells. We now report that calmodulin inhibited Ca2+ release through the IP3 receptor with an IC50 of 4.6 microM if the cytosolic Ca2+ concentration was 0.3 microM or higher. This inhibition was particularly pronounced at low IP3 concentrations. In contrast, calmodulin did not affect IP3-induced Ca2+ release if the cytosolic Ca2+ concentration was below 0.3 microM. Calmodulin also inhibited Ca2+ release through the IP3 receptor in the presence of at least 10 microM Sr2+. We conclude that cytosolic Ca2+ or Sr2+ are absolutely required for the calmodulin-induced inhibition of the IP3-induced Ca2+ release and that this dependence represents the formation of the Ca2+/calmodulin or Sr2+/calmodulin complex.  相似文献   

16.
In non-excitable cells, one major route for Ca2+ influx is through store-operated Ca2+ channels in the plasma membrane. These channels are activated by the emptying of intracellular Ca2+ stores, and in some cell types store-operated influx occurs through Ca2+ release-activated Ca2+ (CRAC) channels. Here, we report that intracellular Ca2+ modulates CRAC channel activity through both positive and negative feedback steps in RBL-1 cells. Under conditions in which cytoplasmic Ca2+ concentration can fluctuate freely, we find that store-operated Ca2+ entry is impaired either following overexpression of a dominant negative calmodulin mutant or following whole-cell dialysis with a calmodulin inhibitory peptide. The peptide had no inhibitory effect when intracellular Ca2+ was buffered strongly at low levels. Hence, Ca2+-calmodulin is not required for the activation of CRAC channels per se but is an important regulator under physiological conditions. We also find that the plasma membrane Ca2+ATPase is the dominant Ca2+ efflux pathway in these cells. Although the activity of the Ca2+ pump is regulated by calmodulin, the store-operated Ca2+ entry is more sensitive to inhibition by the calmodulin mutant than by Ca2+ extrusion. Hence, these two plasmalemmal Ca2+ transport systems may differ in their sensitivities to endogenous calmodulin. Following the activation of Ca2+ entry, the rise in intracellular Ca2+ subsequently feeds back to further inhibit Ca2+ influx. This slow inactivation can be activated by a relatively brief Ca2+ influx (30-60 s); it reverses slowly and is not altered by overexpression of the calmodulin mutant. Hence, the same messenger, intracellular Ca2+, can both facilitate and inactivate Ca2+ entry through store-operated CRAC channels and through different mechanisms.  相似文献   

17.
Initiation of smooth muscle contraction is associated with Ca2+/calmodulin activation of myosin light chain kinase which catalyzes the phosphorylation of the 20-kDa light chain of myosin. In tracheal smooth muscle cells in culture, the extent of myosin light chain phosphorylation is less than 10% at basal cytosolic free Ca2+ concentrations of 150 nM. Stimulation of these cells with serotonin, histamine, carbachol, or the Ca2+ ionophore, ionomycin, increases free cytosolic Ca2+ concentrations and the extent of myosin light chain phosphorylation. Light chain phosphorylation reaches a maximal value of 67% at Ca2+ concentrations below 1 microM. The relationship between the extent of light chain phosphorylation and cytosolic free Ca2+ concentration is apparently independent of the source of free intracellular Ca2+ or the agent used to stimulate the cells and is not altered by pre-exposure of the contractile apparatus to high concentrations of free Ca2+. Pretreatment of cells with 8-bromo-cyclic GMP or forskolin decreases free cytosolic Ca2+ concentrations and the extent of myosin light chain phosphorylation in response to histamine or ionomycin. Pretreatment with 8-bromo-cyclic GMP also decreases the maximal extent of light chain phosphorylation. These results indicate that cytosolic free Ca2+ concentration, per se, is a primary determinant for myosin light chain phosphorylation in tracheal smooth muscle cells.  相似文献   

18.
We measured the concentration of calmodulin required to reverse inhibition by caldesmon of actin-activated myosin MgATPase activity, in a model smooth-muscle thin-filament system, reconstituted in vitro from purified vascular smooth-muscle actin, tropomyosin and caldesmon. At 37 degrees C in buffer containing 120 mM-KCl, 4 microM-Ca2+-calmodulin produced a half-maximal reversal of caldesmon inhibition, but more than 300 microM-Ca2+-calmodulin was necessary at 25 degrees C in buffer containing 60 mM-KCl. The binding affinity (K) of caldesmon for Ca2+-calmodulin was measured by a fluorescence-polarization method: K = 2.7 x 10(6) M-1 at 25 degrees C (60 mM-KCl); K = 1.4 x 10(6) M-1 at 37 degrees C in 70 mM-KCl-containing buffer; K = 0.35 x 10(6) M-1 at 37 degrees C in 120 mM-KCl- containing buffer (pH 7.0). At 37 degrees C/120 mM-KCl, but not at 25 degrees C/60 mM-KCl, Ca2+-calmodulin bound to caldesmon bound to actin-tropomyosin (K = 2.9 x 10(6) M-1). Ca2+ regulation in this system does not depend on a simple competition between Ca2+-calmodulin and actin for binding to caldesmon. Under conditions (37 degrees C/120 mM-KCl) where physiologically realistic concentrations of calmodulin can Ca2+-regulate synthetic thin filaments, Ca2+-calmodulin reverses caldesmon inhibition of actomyosin ATPase by forming a non-inhibited complex of Ca2+-calmodulin-caldesmon-(actin-tropomyosin).  相似文献   

19.
An ATP-dependent transport system which is active at concentrations of free Ca2+ in the submicromolar range has been identified in adipocyte plasma membranes. The system appears to represent the functional component of the high affinity insulin-sensitive calcium-stimulated, magnesium-dependent adenosine triphosphatase preveiously described in the same preparation (Pershadsingh, H. A., and McDonald, J. M. (1979) Nature 281, 495-497). This ATP-dependent Ca2+ transport pump was stimulated approximately 3-fold by the Ca2+-dependent regulatory protein, calmodulin. This effect was confined to the plasma membrane since a similar effect was undetectable in the fraction enriched in endoplasmic reticulum. Calmodulin stimulation was dose-dependent but saturable with half-maximal activation occurring at 0.72 microgram/ml (43 nM). Calmodulin appeared to stimulate the system primarily by decreasing the apparent half-maximal saturation constant for free Ca2+ from 0.20 +/- 0.04 microM to 0.07 +/- 0.01 microM (n = 3). The Hill coefficient increased from 1.6 +/- 0.2 to 3.2 +/- 0.6 (n = 3), thus showing an increased positive cooperativity which allows the pump to be activated by an exceedingly narrow Ca2+ threshold in the presence of calmodulin. The calmodulin stimulation of the plasma membrane Ca2+ extrusion pump in adipocytes, working in opposition to metabolic signals which increase cytoplasmic Ca2+, could constitute a self-regulating negative feedback device for maintaining a low steady state level of intracellular Ca2+. This feedback system may be of critical importance in regulation of cellular metabolism by insulin.  相似文献   

20.
The Ca2+-dependent regulation of human platelet membrane adenylate cyclase has been studied. This enzyme exhibited a biphasic response to Ca2+ within a narrow range of Ca2+ concentrations (0.1-1.0 microM). At low Ca2+ (0.08-0.3 microM) adenylate cyclase was stimulated (Ka = 0.10 microM), whereas at higher Ca2+ (greater than 0.3 microM) the enzyme was inhibited to 70-80% control (Ki = 0.8 microM). Membrane fractions, prepared by washing in the presence of LaCl3 to remove endogenous calmodulin (approximately equal to 70-80% depletion), exhibited no stimulation of adenylate cyclase by Ca2+ but did show the inhibitory phase (Ki = 0.4 microM). The activation phase could be restored to La3+-washed membranes by addition of calmodulin (Ka = 3.0 nM). Under these conditions it was apparent that calmodulin reduced the sensitivity of adenylate cyclase to Ca2+ (Ki = 0.8 microM). Prostaglandin E1 (PGE1) did not alter Ki or Ka values for Ca2+. Calmodulin did not alter the EC50 for PGE1 stimulation of adenylate cyclase but increased the Vmax (1.5-fold). The calmodulin antagonist trifluoperazine potently inhibited adenylate cyclase in native membranes (80%) and to a much lesser extent in La3+-washed membranes (15%). This inhibition was due to interaction of trifluoperazine with endogenous calmodulin since trifluoperazine competitively antagonized the stimulatory effect of calmodulin on adenylate cyclase in La3+-washed membranes. We propose that biphasic Ca2+ regulation of platelet adenylate cyclase functions to both dampen (low Ca2+) and facilitate (high Ca2+) the haemostatic function of platelets.  相似文献   

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