Differential binding of calmodulin domains to constitutive and inducible nitric oxide synthase enzymes

Calmodulin (CaM) is a Ca2+ signal transducing protein that binds and activates many cellular enzymes with physiological relevance, including the mammalian nitric oxide synthase (NOS) isozymes: endothelial NOS (eNOS), neuronal NOS (nNOS), and inducible NOS (iNOS). The mechanism of CaM binding and activation to the iNOS enzyme is poorly understood in part due to the strength of the bound complex and the difficulty of assessing the role played by regions outside of the CaM-binding domain. To further elucidate these processes, we have developed the methodology to investigate CaM binding to the iNOS holoenzyme and generate CaM mutant proteins selectively labeled with fluorescent dyes at specific residues in the N-terminal lobe, C-terminal lobe, or linker region of the protein. In the present study, an iNOS CaM coexpression system allowed for the investigation of CaM binding to the holoenzyme; three different mutant CaM proteins with cysteine substitutions at residues T34 (N-domain), K75 (central linker), and T110 (C-domain) were fluorescently labeled with acrylodan or Alexa Fluor 546 C5-maleimide. These proteins were used to investigate the differential association of each region of CaM with the three NOS isoforms. We have also N-terminally labeled an iNOS CaM-binding domain peptide with dabsyl chloride in order to perform FRET studies between Alexa-labeled residues in the N- and C-terminal domains of CaM to determine CaM's orientation when associated to iNOS. Our FRET results show that CaM binds to the iNOS CaM-binding domain in an antiparallel orientation. Our steady-state fluorescence and circular dichroism studies show that both the N- and C-terminal EF hand pairs of CaM bind to the CaM-binding domain peptide of iNOS in a Ca2+-independent manner; however, only the C-terminal domain showed large Ca2+-dependent conformational changes when associated with the target sequence. Steady-state fluorescence showed that Alexa-labeled CaM proteins are capable of binding to holo-iNOS coexpressed with nCaM, but this complex is a transient species and can be displaced with the addition of excess CaM. Our results show that CaM does not bind to iNOS in a sequential manner as previously proposed for the nNOS enzyme. This investigation provides additional insight into why iNOS remains active even under basal levels of Ca2+ in the cell.     

Nitric-oxide synthase (NOS) reductase domain models suggest a new control element in endothelial NOS that attenuates calmodulin-dependent activity

Inducible (iNOS) and constitutive (eNOS, nNOS) nitric-oxide synthases differ in their Ca2+-calmodulin (CaM) dependence. iNOS binds CaM irreversibly but eNOS and nNOS, which bind CaM reversibly, have inserts in their reductase domains that regulate electron transfer. These include the 43-45-amino acid autoinhibitory element (AI) that attenuates electron transfer in the absence of CaM, and the C-terminal 20-40-amino acid tail that attenuates electron transfer in a CaM-independent manner. We constructed models of the reductase domains of the three NOS isoforms to predict the structural basis for CaM-dependent regulation. We have identified and characterized a loop (CD2A) within the NOS connecting domain that is highly conserved by isoform and that, like the AI element, is within direct interaction distance of the CaM binding region. The eNOS CD2A loop (eCD2A) has the sequence 834KGSPGGPPPG843, and is truncated to 809ESGSY813 (iCD2A) in iNOS. The eCD2A contributes to the Ca2+ dependence of CaM-bound activity to a level similar to that of the AI element. The eCD2A plays an autoinhibitory role in the control of NO, and CaM-dependent and -independent reductase activity, but this autoinhibitory function is masked by the dominant AI element. Finally, the iCD2A is involved in determining the salt dependence of NO activity at a post-flavin reduction level. Electrostatic interactions between the CD2A loop and the CaM-binding region, and CaM itself, provide a structural means for the CD2A to mediate CaM regulation of intra-subunit electron transfer within the active NOS complex.     

Calmodulin-dependent adenylate cyclase activity in rat cerebral cortex: effects of divalent cations, forskolin and isoprenaline

The role of calcium-calmodulin (Ca2+-CaM) in the modulation of beta-adrenergic adenylate cyclase activity in rat cerebral cortex has been studied. In addition, the effects of manganese (Mn2+) and forskolin on CaM-dependent enzyme activity were investigated. At 2 mM magnesium (Mg2+) low concentrations of Ca2+ stimulated the enzyme activity (Ka 0.25 +/- 0.08 microM), whereas higher Ca2+ levels (greater than 2 microM) inhibited the activity. No activating effect of Ca2+ was observed in CaM-depleted membranes, but the inhibitory effect persisted and the stimulatory action of Ca2+ could be restored by addition of exogenous CaM. The ability of Ca2+ to activate the enzyme was reduced by increasing concentrations of Mg2+. At 10 mM Mg2+ the apparent Ka of Ca2+ was 0.55 +/- 0.16 microM and half-maximal inhibition was observed at 80-120 microM Ca2+. A synergistic effect was observed between Ca2+ and isoprenaline on the adenylate cyclase activity. Calcium did not alter the apparent Ka of isoprenaline (0.9 +/- 0.27 microM) and isoprenaline did not change the apparent Ka of Ca2+. However, isoprenaline decreased the apparent Ka of CaM; 0.11 +/- 0.07 micrograms vs. 0.32 +/- 0.1 micrograms (0.5 ml assay mixture)-1, with and without isoprenaline, respectively. A synergistic effect was also observed between Ca2+ and forskolin, but no change in their apparent Ka values was found. Furthermore, Mn2+ was found to activate the enzyme through CaM. These data demonstrate that Ca2+ -CaM potentiates beta-adrenergic adenylate cyclase activity and thus is able to modulate neurotransmitter stimulation in cortex. Furthermore, both forskolin and Mn2+ affect CaM-dependent enzyme activity. Forskolin potentiates Ca2+-CaM stimulation, while Mn2+ increases the activity by activating the enzyme through CaM.     

Structural elements contribute to the calcium/calmodulin dependence on enzyme activation in human endothelial nitric-oxide synthase

Two regions, located at residues 594-606/614-645 and residues 1165-1178, are present in the reductase domain of human endothelial nitric-oxide synthase (eNOS) but absent in its counterpart, inducible nitric-oxide synthase (iNOS). We previously demonstrated that removing residues 594-606/614-645 resulted in an enzyme (Delta45) containing an intrinsic calmodulin (CaM) purified from an Sf9/baculovirus expression system (Chen, P.-F., and Wu, K.K. (2000) J. Biol. Chem. 275, 13155-13163). Here we have further elucidated the differential requirement of Ca2+/CaM for enzyme activation between eNOS and iNOS by either deletion of residues 1165-1178 (Delta14) or combined deletions of residues 594-606/614-645 and 1165-1178 (Delta45/ Delta14) from eNOS to mimic iNOS. We measured the catalytic rates using purified proteins completely free of CaM. Steady-state analysis indicated that the Delta45 supported NO synthesis in the absence of CaM at 60% of the rate in its presence, consistent with our prior result that CaM-bound Delta45 retained 60% of its activity in the presence of 10 mm EGTA. Mutant Delta14 displayed a 1.5-fold reduction of EC50 for Ca2+/CaM-dependence in l-citrulline formation, and a 2-4-fold increase in the rates of NO synthesis, NADPH oxidation, and cytochrome c reduction relative to the wild type. The basal rates of double mutant Delta45/Delta14 in NO production, NADPH oxidation, and cytochrome c reduction were 3-fold greater than those of CaM-stimulated wild-type eNOS. Interestingly, all three activities of Delta45/ Delta14 were suppressed rather than enhanced by Ca2+/CaM, indicating a complete Ca2+/CaM independence for those reactions. The results suggest that the Ca2+/CaM-dependent catalytic activity of eNOS appears to be conferred mainly by these two structural elements, and the interdomain electron transfer from reductase to oxygenase domain does not require Ca2+/CaM when eNOS lacks these two segments.     

Novel aspects of calmodulin target recognition and activation.

Several crystal and NMR structures of calmodulin (CaM) in complex with fragments derived from CaM-regulated proteins have been reported recently and reveal novel ways for CaM to interact with its targets. This review will discuss and compare features of the interaction between CaM and its target domains derived from the plasma membrane Ca2+-pump, the Ca2+-activated K+-channel, the Ca2+/CaM-dependent kinase kinase and the anthrax exotoxin. Unexpected aspects of CaM/target interaction observed in these complexes include: (a) binding of the Ca2+-pump domain to only the C-terminal part of CaM (b) dimer formation with fragments of the K+-channel (c) insertion of CaM between two domains of the anthrax exotoxin (d) binding of Ca2+ ions to only one EF-hand pair and (e) binding of CaM in an extended conformation to some of its targets. The mode of interaction between CaM and these targets differs from binding conformations previously observed between CaM and peptides derived from myosin light chain kinase (MLCK) and CaM-dependent kinase IIalpha (CaMKIIalpha). In the latter complexes, CaM engulfs the CaM-binding domain peptide with its two Ca2+-binding lobes and forms a compact, ellipsoid-like complex. In the early 1990s, a model for the activation of CaM-regulated proteins was developed based on this observation and postulated activation through the displacement of an autoinhibitory or regulatory domain from the target protein upon binding of CaM. The novel structures of CaM-target complexes discussed here demonstrate that this mechanism of activation may be less general than previously believed and seems to be not valid for the anthrax exotoxin, the CaM-regulated K+-channel and possibly also not for the Ca2+-pump.     

Differential activation of nitric-oxide synthase isozymes by calmodulin-troponin C chimeras

The interactions of neuronal nitric-oxide synthase (nNOS) with calmodulin (CaM) and mutant forms of CaM, including CaM-troponin C chimeras, have been previously reported, but there has been no comparable investigation of CaM interactions with the other constitutively expressed NOS (cNOS), endothelial NOS (eNOS), or the inducible isoform (iNOS). The present study was designed to evaluate the role of the four CaM EF hands in the activation of eNOS and iNOS. To assess the role of CaM regions on aspects of enzymatic function, three distinct activities associated with NOS were measured: NADPH oxidation, cytochrome c reduction, and nitric oxide (*NO) generation as assessed by the oxyhemoglobin capture assay. CaM activates the cNOS enzymes by a mechanism other than stimulating electron transfer into the oxygenase domain. Interactions with the reductase moiety are dominant in cNOS activation, and EF hand 1 is critical for activation of both nNOS and eNOS. Although the activation patterns for nNOS and eNOS are clearly related, effects of the chimeras on all the reactions are not equivalent. We propose that cytochrome c reduction is a measure of the release of the FMN domain from the reductase complex. In contrast, cytochrome c reduction by iNOS is readily activated by each of the chimeras examined here and may be constitutive. Each of the chimeras were co-expressed with the human iNOS enzyme in Escherichia coli and subsequently purified. Domains 2 and 3 of CaM contain important elements required for the Ca2+/CaM independence of *NO production by the iNOS enzyme. The disparity between cytochrome c reduction and *NO production at low calcium can be attributed to poor association of heme and FMN domains when the bound CaM constructs are depleted of Ca2+. In general cNOSs are much more difficult to activate than iNOS, which can be attributed to their extra sequence elements, which are adjacent to the CaM-binding site and associated with CaM control.     

Purified red blood cell Ca2+-pump ATPase: evidence for direct inhibition by presumed anti-calmodulin drugs in the absence of calmodulin

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.     

Binding and activation of nitric oxide synthase isozymes by calmodulin EF hand pairs

Calmodulin (CaM) is a cytosolic Ca(2+) signal-transducing protein that binds and activates many different cellular enzymes with physiological relevance, including the nitric oxide synthase (NOS) isozymes. CaM consists of two globular domains joined by a central linker; each domain contains an EF hand pair. Four different mutant CaM proteins were used to investigate the role of the two CaM EF hand pairs in the binding and activation of the mammalian inducible NOS (iNOS) and the constitutive NOS (cNOS) enzymes, endothelial NOS (eNOS) and neuronal NOS (nNOS). The role of the CaM EF hand pairs in different aspects of NOS enzymatic function was monitored using three assays that monitor electron transfer within a NOS homodimer. Gel filtration studies were used to determine the effect of Ca(2+) on the dimerization of iNOS when coexpressed with CaM and the mutant CaM proteins. Gel mobility shift assays were performed to determine binding stoichiometries of CaM proteins to synthetic NOS CaM-binding domain peptides. Our results show that the N-terminal EF hand pair of CaM contains important binding and activating elements for iNOS, whereas the N-terminal EF hand pair in conjunction with the central linker region is required for cNOS enzyme binding and activation. The iNOS enzyme must be coexpressed with wild-type CaM in vitro because of its propensity to aggregate when residues of the highly hydrophobic CaM-binding domain are exposed to an aqueous environment. A possible role for iNOS aggregation in vivo is also discussed.     

The effect of Ca2+ and calmodulin on the inhibition of Ca2(+)+Mg2(+)-ATPase in erythrocyte ghost membranes by nonpolar and polar carbodiimides

N,N'-dicyclohexylcarbodiimide (DCCD) and 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide (CMCD) inhibited calmodulin-dependent Ca2(+)+Mg2(+)-ATPase activity in erythrocyte ghost membranes. The extent of the inhibition caused by carbodiimides strongly depended on their hydrophobicity. Hydrophobic DCCD was a more potent inhibitor then hydrophilic CMCD. Calmodulin (CaM) protected the enzyme against the former carbodiimide, whereas Ca2+ did the same against the latter. In contrast to previous observations made by Villalobo et al., on the purified enzyme, neither carbodiimide affected the calmodulin-independent ATPase activity in ghost membranes. Inhibition of the calmodulin-dependent ATPase activity was due to a decrease of the maximum activity, whereas the Km value for Ca2+ remained unchanged. Titration of erythrocyte ghost membranes with CaM revealed a biphasic response of ATPase to this activator. Two affinity constants were found for CaM, 0.64 nM and 14 nM. DCCD affected the interaction with CaM at high- and low-affinity binding sites in a competitive manner. CMCD acted as a noncompetitive inhibitor for CaM low-affinity sites, whereas it behaved in a competitive way against CaM interaction with high-affinity sites. In E2 form (stabilized by vanadate and EGTA) ATPase was more sensitive to carbodiimides than in E1 form (induced by La3+).     

Calcium-deficient calmodulin binding and activation of neuronal and inducible nitric oxide synthases

The nitric oxide synthase (NOS) enzymes are bound and activated by the Ca(2+)-binding protein, calmodulin (CaM). We have utilized CaM mutants deficient in binding Ca(2+) with mutations in the N-lobe (CaM(12)), the C-lobe (CaM(34)), or both lobes of CaM (CaM(1234)) to determine their effect on the binding and activation of the Ca(2+)-dependent neuronal (nNOS) and Ca(2+)-independent inducible NOS (iNOS) isoforms. Four different kinetic assays were employed to monitor the effect of these CaM mutants on electron transfer rates in NOS. Protein-protein interactions between CaM and NOS were studied using steady-state fluorescence and spectropolarimetry to monitor the binding of these CaM mutants to nNOS and iNOS CaM-binding domain peptides. The CaM mutants were unable to activate nNOS, however, our CD results show that the C-terminal lobe of CaM is capable of binding to nNOS peptide in the presence of Ca(2+). Our results prove for the first time without the use of chelators that apo-CaM is capable of binding to iNOS peptides and holoenzymes.     

Saccharomyces cerevisiae protein kinase dependent on Ca2+ and calmodulin.

A Ca2+- and calmodulin (CaM)-dependent protein kinase of Saccharomyces cerevisiae was partially purified by CaM affinity chromatography of the soluble fraction, and the properties of the enzyme were investigated. The protein kinase activity of the affinity-purified preparation was stimulated at least eightfold by the simultaneous presence of Ca2+ and CaM. The enzyme stimulation was strongly inhibited by trifluoperazine (TFP), a CaM antagonist. When the kinase was incubated in the presence of ATP, Ca2+, and CaM before the assay, the enzyme showed activity even in the presence of the Ca2+ chelator ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) and TFP. The conversion to this Ca2+- and CaM-independent form occurred very rapidly under the incubation conditions required for protein phosphorylation by the kinase. At the highest level of conversion, Ca2+- and CaM-independent kinase activity, which was measured in the presence of EGTA and TFP, was nearly equal to the total kinase activity, which was measured in the presence of Ca2+ and CaM. A protein with a molecular weight of 58,000 was the major species that was phosphorylated in a Ca2+- and CaM-dependent manner by incubation of the CaM affinity-purified proteins with [gamma-32P]ATP. The protein kinase activity of the protein with the same molecular weight was demonstrated by in situ protein phosphorylation in sodium dodecyl sulfate-polyacrylamide gels by using casein as the substrate, after removal of the detergent from electrophoresed CaM-binding proteins. These data indicate that phosphorylation of the kinase is responsible for the conversion of enzyme activity. Enzyme regulation by this mode may play an important role in integrating cellular functions during the cell cycle. A possible role for the Ca2+-and CaM-dependent protein kinase in the signal transduction of the mating pheromone alpha factor is also discussed.     

Circular dichroism and fluorescence studies on interaction of calmodulin (CaM) with purified (Ca2(+)-Mg2+)ATPase of erythrocyte ghosts

It was found, using circular dichroism spectroscopy, that CaM, in the presence of Ca2+, decreases the alpha-helix content of (Ca2(+)-Mg2+)ATPase of porcine erythrocytes from 66% to 55%. In the absence of Ca2+ the enzyme showed 46% of alpha-helix. Moreover, quenching of the ATPase intrinsic fluorescence by acrylamide indicated that, depending on the enzyme conformational status, the accessibility of its tryptophan residues is influenced by direct interaction with CaM at micromolar Ca2+ concentration. This was also confirmed by the observation that fluorescence energy transfer occurred from tryptophan residues of (Ca2(+)-Mg2+)ATPase to dansylated CaM. The presented results may indicate that binding of CaM gives rise to a novel conformational state of the enzyme, distinct from E1 and E2 forms of the Ca2+ pump.     

Calmodulin activates inositol 1,4,5-trisphosphate 3-kinase activity in pig aortic smooth muscle.

The effects of calmodulin (CaM) on inositol 1,4,5-trisphosphate (InsP3) 3-kinase activity in pig aortic smooth muscle were examined. The cytosol fraction of muscle cells, containing 1.2-2.0 micrograms of CaM/mg of cytosol protein (thus 0.12-0.2%, w/w), showed a Ca2+-dependent InsP3 3-kinase activity, and there was no further activation by exogenous addition of CaM purified from dog brain. (NH4)2SO4 fractionation of the cytosol fraction revealed that a 20-60%-satd.-(NH4)2SO4 fraction was rich in the enzyme activity, and the activity without exogenous CaM was still dependent on Ca2+, although the CaM content in this fraction was minute (0.013-0.016%, w/w). The kinase activity observed in the absence of exogenous CaM became insensitive to Ca2+ when a 20-60%-satd.-(NH4)2SO4 fraction was applied to a DEAE-cellulose column, but exogenous addition of CaM increased the enzyme activity from 80-120 to 450 pmol/min per mg of protein, with addition of 10 microM free Ca2+. A fraction separated by DEAE-cellulose chromatography was applied to a CaM affinity column. The kinase activity was retained on the column in the presence of Ca2+, and was eluted by lowering the free Ca2+ concentration by adding EGTA. These results directly show that CaM activates InsP3 3-kinase activity and the enzyme becomes sensitive to Ca2+.     

果蝇TRP离子通道C端一个新钙调蛋白结合位点的鉴定和分析

瞬时受体电位(TRP)通道是一类钙离子透过性的阳离子通道蛋白家族,参与了视觉、味觉、温度感受等重要的生物学过程。之前的研究表明,钙离子既能够正反馈也能够负反馈地调节瞬时受体电位通道的活性,而这种调节可能是通过钙调蛋白（calmodulin,CaM）与TRP通道的相互作用来进行的。为了阐明这一调控机制,我们首先需要对钙调蛋白与瞬时受体电位通道之间的相互作用进行详细的生化研究。在此项研究中,通过大肠杆菌表达系统,表达和纯化了果蝇瞬时受体电位通道羧基末端不同长短的蛋白片段,并发现了一个新的钙调蛋白结合位点。通过快速蛋白液相色谱、静态光散射以及等温量热滴定技术,鉴定了这一钙调蛋白结合位点与果蝇瞬时受体电位通道之间的相互作用,发现它们在钙离子依赖的条件下,可以形成亲和力非常强的稳定的蛋白复合物（解离常数在01～1微摩尔范围）。此外,通过合成多肽的方法,鉴定了果蝇瞬时受体电位通道913～939片段为该钙调蛋白结合位点的核心区域。最后,通过突变实验,进一步明确了果蝇瞬时受体电位通道922位的酪氨酸以及923位的缬氨酸为其钙调蛋白结合位点的关键氨基酸。总而言之,本研究发现和鉴定了果蝇瞬时受体电位通道上一个新的钙依赖的钙调蛋白结合位点,这一发现将为研究瞬时受体电位通道的体内功能提供生化基础,为阐明钙离子通过钙调蛋白调节瞬时受体电位通道的分子机制做出贡献。     

Calcium-binding sites of calmodulin and electron transfer by inducible nitric oxide synthase

Like that of the neuronal nitric oxide synthase (nNOS), the binding of Ca(2+)-bound calmodulin (CaM) also regulates the activity of the inducible isoform (iNOS). However, the role of each of the four Ca(2+)-binding sites of CaM in the activity of iNOS is unclear. Using a series of single-point mutants of Drosophila melanogaster CaM, the effect that mutating each of the Ca(2+)-binding sites plays in the transfer of electrons within iNOS has been examined. The same Glu (E) to Gln (Q) mutant series of CaM used previously [Stevens-Truss, R., Beckingham, K., and Marletta, M. A. (1997) Biochemistry 36, 12337-12345] to study the role of the Ca(2+)-binding sites in the activity of nNOS was used for these studies. We demonstrate here that activity of iNOS is dependent on Ca(2+) being bound to sites II (B2Q) and III (B3Q) of CaM. Nitric oxide ((*)NO) producing activity (as measured using the hemoglobin assay) of iNOS bound to the B2Q and B3Q CaMs was found to be 41 and 43% of the wild-type activity, respectively. The site I (B1Q) and site IV (B4Q) CaM mutants only minimally affected (*)NO production (95 and 90% of wild-type activity, respectively). These results suggest that NOS isoforms, although all possessing a prototypical CaM binding sequence and requiring CaM for activity, interact with CaM differently. Moreover, iNOS activation by CaM, like nNOS, is not dependent on Ca(2+) being bound to all four Ca(2+)-binding sites, but has specific and distinct requirements. This novel information, in addition to helping us understand NOS, should aid in our understanding of CaM target activation.     

Calcium dependence of the interaction between calmodulin and anthrax edema factor

Edema factor (EF), a toxin from Bacillus anthracis (anthrax), possesses adenylyl cyclase activity and requires the ubiquitous Ca2+-sensor calmodulin (CaM) for activity. CaM can exist in three major structural states: an apo state with no Ca2+ bound, a two Ca2+ state with its C-terminal domain Ca2+-loaded, and a four Ca2+ state in which the lower Ca2+ affinity N-terminal domain is also ligated. Here, the interaction of EF with the three Ca2+ states of CaM has been examined by NMR spectroscopy and changes in the Ca2+ affinity of CaM in the presence of EF have been determined by flow dialysis. Backbone chemical shift perturbations of CaM show that EF interacts weakly with the N-terminal domain of apoCaM. The C-terminal CaM domain only engages in the interaction upon Ca2+ ligation, rendering the overall interaction much tighter. In the presence of EF, the C-terminal domain binds Ca2+ with higher affinity, but loses binding cooperativity, whereas the N-terminal domain exhibits strongly reduced Ca2+ affinity. As judged by chemical shift differences, the N-terminal CaM domain remains bound to EF upon subsequent Ca2+ ligation. This Ca2+ dependence of the EF-CaM interaction differs from that observed for most other CaM targets, which normally interact only with the Ca2+-bound CaM domains and become active following the transition to the four Ca2+ state.     

重金属离子对猪红细胞膜Ca~(2+)-ATP酶的作用

猪红细胞膜Ca~(2+)-ATP酶是一种钙调蛋白(CaM)依赖酶,其活力又依赖巯基的完整性。实验应用Ca~(2+)-ATP酶这一模型体系观察到重金属离子,Pb~(2+)、Cd~(2+)和Hg~(2+)都能替代Ca~(2+),激活CaM,从而激活Ca~(2+)-ATP酶;其最大刺激活力分别为85％、80％和30％,半刺激浓度分别为32、27和0.7μmol/L。当三种重金属离子的浓度增加时,则与Ca~(2+)-ATP酶的巯基结合,抑制酶的活力,Pb2~(2+)、Cd~(2+)和Hg~(2+)的半抑制浓度分别为370、440和2μmol/L。抑制作用为渐进性过程,而刺激作用为即时效应。抑制作用可为巯基化物,特别是二巯基化物所逆转。研究结果提示,CaM可能是重金属中毒最初作用的靶分子,而重金属中毒不仅使CaM“开关”失灵,还可能导致细胞内Ca~(2+)的调节全面失控。     

Conformational changes of (Ca2+-Mg2+)-ATPase of erythrocyte plasma membrane caused by calmodulin and phosphatidylserine as revealed by circular dichroism and fluorescence studies

Two spectroscopic techniques, circular dichroism and steady-state fluorescence, were employed in order to study conformational changes of the purified, detergent-solubilized (Ca2+-Mg2+)-ATPase of porcine erythrocyte ghost membranes. Circular dichroism (CD) spectra in the peptide region were obtained from the purified (Ca2+-Mg2+)-ATPase of porcine erythrocyte ghost membranes with the aim to investigate the secondary structure of the enzyme in the presence of calmodulin (CaM) or phosphatidylserine (PS), as well as in the E1 and E2 states. The E1 conformation was stabilized by 10 microM free Ca2+, while the E2 conformation was stabilized by 0.1 mM ethylene glycol bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). It was found that the E1 and E2 states of the enzyme strikingly differed in their secondary structure (66% and 46% of calculated alpha-helix content, respectively). In the presence of Ca2+, PS decreased the helical content of the ATPase to 61%, while CaM to 55%. Quenching of intrinsic fluorescence of (Ca2+-Mg2+)-ATPase by acrylamide, performed in the presence of Ca2+, gave evidence for a single class of tryptophan residues with Stern-Volmer constant (KSV) of 10 M-1. Accessibility of tryptophan residues varied depending on the conformational status of the enzyme. Addition of PS and CaM decreased the KSV value to 7.6 M-1 and 8.5 M-1, respectively. In the absence of Ca2+, KSV was 7.0 M-1. KI and CsCl were less effective as quenchers. The fluorescence energy transfer between (Ca2+-Mg2+)-ATPase tryptophan residues and dansyl derivative of covalently labeled CaM occurred in the presence of EGTA, but was further promoted by Ca2+. It is concluded that the interaction of CaM and PS with (Ca2+-Mg2+)-ATPase results in different conformational states of the enzyme. CD and fluorescence spectroscopy allowed to distinguish these states from the E1 and E2 conformational forms of the ATPase.     

Interaction between lipids and bovine brain calmodulin: lysophosphatidylcholine-induced conformation change

We have monitored the interaction of several lipids with the bovine brain calmodulin(CaM) and analyzed the effect of lysophosphatidylcholine(lyso-PC, 2-50 micrograms/ml) on conformation of CaM and the interaction between CaM and CaM-binding protein(CaMBP), using a fluorescence signal of 1-(dimethylamino)naphthalene-5-sulfonate-labeled CaM(DNS-CaM). Lyso-PC(egg, 20 micrograms/ml), among various natural lipids including phosphatidylserine(PS), phosphatidylinositol(PI), phosphatidylethanolamine (PE) and their lyso forms, greatly and dose-dependently enhanced the intensity of DNS fluorescence of DNS-CaM in the presence (100 microM CaCl2) and absence (1 mM EGTA) of Ca2+. Apparent dissociation constants calculated from the fluorometric titrations of binding of lyso-PC to DNS-CaM were 0.6 and 3.7 micrograms/ml in the presence and absence of Ca2+, respectively. Lyso-PC remarkably prevented both trypsin-induced quenching of the fluorescence of DNS-CaM and tryptic digestion of native CaM in the absence of Ca2+. Enhancement of DNS fluorescence of DNS-CaM by CaMBP was observed only in the presence of Ca2+ and lyso-PC could further increase the fluorescence intensity of the complex. These all results suggest that lyso-PC can modulate the interaction between CaM and CaMBP as a result of its direct effect on conformation of CaM.