The effect of berbamine derivatives on activated Ca2+-stimulated Mg2+-dependent ATPase in erythrocyte membranes.

Ca2+-stimulated Mg2+-dependent ATPase (Ca2+ + Mg2+-ATPase) stimulated by calmodulin, by partial proteolysis or by oleic acid in erythrocyte membranes was inhibited by various derivatives of the naturally occurring alkaloid berbamine. The ability of these derivatives to inhibit trypsin-activated Ca2+ + Mg2+-ATPase correlated well with their ability to inhibit the calmodulin-stimulated enzyme. Inhibition of the trypsin-activated Ca2+ + Mg2+-ATPase by O-4-(ethoxybutyl)berbamine (EBB) was competitive with respect to ATP. The Ki for inhibition was about 8 microM. These results suggest that the binding site of EBB on the activated Ca2+ + Mg2+-ATPase may bear structural similarity to that on calmodulin, and may be closely related to the ATP-binding site on the enzyme.     

Reversible inhibition of the calcium-pumping ATPase in native cardiac sarcoplasmic reticulum by a calmodulin-binding peptide. Evidence for calmodulin-dependent regulation of the V(max) of calcium transport

Calmodulin (CaM) and Ca(2+)/CaM-dependent protein kinase II (CaM kinase) are tightly associated with cardiac sarcoplasmic reticulum (SR) and are implicated in the regulation of transmembrane Ca(2+) cycling. In order to assess the importance of membrane-associated CaM in modulating the Ca(2+) pump (Ca(2+)-ATPase) function of SR, the present study investigated the effects of a synthetic, high affinity CaM-binding peptide (CaM BP; amino acid sequence, LKWKKLLKLLKKLLKLG) on the ATP-energized Ca(2+) uptake, Ca(2+)-stimulated ATP hydrolysis, and CaM kinase-mediated protein phosphorylation in rabbit cardiac SR vesicles. The results revealed a strong concentration-dependent inhibitory action of CaM BP on Ca(2+) uptake and Ca(2+)-ATPase activities of SR (50% inhibition at approximately 2-3 microM CaM BP). The inhibition, which followed the association of CaM BP with its SR target(s), was of rapid onset (manifested within 30 s) and was accompanied by a decrease in V(max) of Ca(2+) uptake, unaltered K(0.5) for Ca(2+) activation of Ca(2+) transport, and a 10-fold decrease in the apparent affinity of the Ca(2+)-ATPase for its substrate, ATP. Thus, the mechanism of inhibition involved alterations at the catalytic site but not the Ca(2+)-binding sites of the Ca(2+)-ATPase. Endogenous CaM kinase-mediated phosphorylation of Ca(2+)-ATPase, phospholamban, and ryanodine receptor-Ca(2+) release channel was also strongly inhibited by CaM BP. The inhibitory action of CaM BP on SR Ca(2+) pump function and protein phosphorylation was fully reversed by exogenous CaM (1-3 microM). A peptide inhibitor of CaM kinase markedly attenuated the ability of CaM to reverse CaM BP-mediated inhibition of Ca(2+) transport. These findings suggest a critical role for membrane-bound CaM in controlling the velocity of Ca(2+) pumping in native cardiac SR. Consistent with its ability to inhibit SR Ca(2+) pump function, CaM BP (1-2.5 microM) caused marked depression of contractility and diastolic dysfunction in isolated perfused, spontaneously beating rabbit heart preparations. Full or partial recovery of contractile function occurred gradually following withdrawal of CaM BP from the perfusate, presumably due to slow dissociation of CaM BP from its target sites promoted by endogenous cytosolic CaM.     

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.     

Association between human erythrocyte calmodulin and the cytoplasmic surface of human erythrocyte membranes

This report describes Ca2+-dependent binding of 125I-labeled calmodulin (125I-CaM) to erythrocyte membranes and identification of two new CaM-binding proteins. Erythrocyte CaM labeled with 125I-Bolton Hunter reagent fully activated erythrocyte (Ca2+ + Mg2+)-ATPase. 125I-CaM bound to CaM depleted membranes in a Ca2+-dependent manner with a Ka of 6 x 10(-8) M Ca2+ and maximum binding at 4 x 10(-7) M Ca2+. Only the cytoplasmic surface of the membrane bound 125I-CaM. Binding was inhibited by unlabeled CaM and by trifluoperazine. Reduction of the free Ca2+ concentration or addition of trifluoperazine caused a slow reversal of binding. Nanomolar 125I-CaM required several hours to reach binding equilibrium, but the rate was much faster at higher concentrations. Scatchard plots of binding were curvilinear, and a class of high affinity sites was identified with a KD of 0.5 nM and estimated capacity of 400 sites per cell equivalent for inside-out vesicles (IOVs). The high affinity sites of IOVs most likely correspond to Ca2+ transporter since: (a) Ka of activation of (Ca2+ + Mg2+)-ATPase and KD for binding were nearly identical, and (b) partial digestion of IOVs with alpha-chymotrypsin produced activation of the (Ca2+ + Mg2+)-ATPase with loss of the high affinity sites. 125I-CaM bound in solution to a class of binding proteins (KD approximately 55 nM, 7.3 pmol per mg of ghost protein) which were extracted from ghosts by low ionic strength incubation. Soluble binding proteins were covalently cross-linked to 125I-CaM with Lomant's reagent, and 2 bands of 8,000 and 40,000 Mr (Mr of CaM subtracted) and spectrin dimer were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiography. The 8,000 and 40,000 Mr proteins represent a previously unrecognized class of CaM-binding sites which may mediate unexplained Ca2+-induced effects in the erythrocyte.     

Inhibition of Ca2+-dependent protein kinase and Ca2+/Mg2+-ATPase in cardiac sarcolemma by the anti-calmodulin drug calmidazolium

Sarcolemma (SL) vesicles, isolated from pig heart, contain both a Ca2+-calmodulin-dependent protein kinase (CaM-PK) and a Ca2+-dependent Mg2+-ATPase (Ca2+/Mg2+)-ATPase). Some of their properties have been compared: their affinity for Ca2+ ions, dependence on exogenous calmodulin (CaM) and sensitivity to the anti-CaM drug calmidazolium (R24571). The properties of Ca2+-CaM-dependent brain phosphodiesterase (PDE) have also been examined. R24571 appeared to be the most potent inhibitor from brain PDE. For the three enzymes studied, exogenously added CaM was able to antagonize the R24571 inhibition, although the efficiency to counteract was rather low in the case of the SL Ca2+/Mg2+-ATPase. R24571 decreased the affinity of the Ca2+/Mg2+-ATPase for Ca2+ ions (KCa 0.35 versus 0.75 microM) and exerted an inhibition non-competitive with Ca2+ ions on the other CaM-dependent enzymes. Membrane-bound CaM, which is involved in controlling the Ca2+/Mg2+-ATPase, appeared to be present in a stoichiometry varying from 1:1 to 1:4 compared to the 32P-intermediate of the ATPase. R24571 treatment of SL vesicles selectively solubilized a number of proteins in the molecular range of 13-20 kD, which may include CaM. The results suggest that different mechanisms are involved in the CaM control of the two SL enzymes studied.     

粉防已碱与红细胞膜ATPase相互作用的研究

粉防已碱是一种新的钙调蛋白拮抗剂,专一性抑制人红细胞膜上依赖CaM的Ca~(2+)-Mg~(2+)-ATPase。在较高浓度下,它也不同程度地抑制Ca~(2+)-Mg~(2+)-ATPase基本活性、Na~+-K~+-ATPase和Mg~(2+)-ATPase的活性。 除CaM外,不饱和脂肪酸和有限水解均导致膜Ca~(2+)-Mg~(2+)-ATPase的活化,所有这些活化作用被Tet在大约相同的浓度范围内抑制,表明Tet除与CaM结合外,也与膜Ca~(2+)-Mg~(2+)-ATPase结合。 Tet具有抗抵渗溶血的性能,反映了拮抗CaM与药物的膜稳定性间存在相关性。     

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.     

Regulation of the RYR1 and RYR2 Ca2+ release channel isoforms by Ca2+-insensitive mutants of calmodulin

Calmodulin (CaM) may function as a regulatory subunit of ryanodine receptor (RYR) channels, modulating both channel activation and inhibition by Ca2+; however, mechanisms underlying differences in CaM regulation of the RYR isoforms expressed in skeletal muscle (RYR1) and cardiac muscle (RYR2) are poorly understood. Here we use a series of CaM mutants deficient in Ca2+ binding to compare determinants of CaM regulation of the RYR1 and RYR2 isoforms. In submicromolar Ca2+, activation of the RYR1 isoform by each of the single-point CaM mutants was similar to that by wild-type apoCaM, whereas in micromolar Ca2+, RYR1 inhibition by Ca2+CaM was abolished by mutations targeting CaM's C-terminal Ca2+ sites. In contrast to the RYR1, no activation of the cardiac RYR2 isoform by wild-type CaM was observed, but rather CaM inhibited the RYR2 at all Ca2+ concentrations (100 nM to 1 mM). Consequently, whereas the apparent Ca2+ sensitivity of the RYR1 isoform was enhanced in the presence of CaM, the RYR2 displayed the opposite response (RYR2 Ca2+ EC50 increased 7-10-fold in the presence of 5 microM wild-type CaM). CaM inhibition of the RYR2 was nonetheless abolished by each of four mutations targeting individual CaM Ca2+ sites. Furthermore, a mutant CaM deficient in Ca2+ binding at all four Ca2+ sites significantly activated the RYR2 and acted as a competitive inhibitor of RYR2 regulation by wild-type Ca2+CaM. We conclude that Ca2+ binding to CaM determines the effect of CaM on both RYR1 and RYR2 channels and that isoform differences in CaM regulation reflect the differential tuning of Ca2+ binding sites on CaM when bound to the different RYRs. These results thus suggest a novel mechanism by which CaM may contribute to functional diversity among the RYR isoforms.     

稀土离子对CaM及Ca2+-Mg2+-ATPase活力及CD研究

研究了稀土离子（Ｌｎ３＋）对钙调蛋白（ＣａＭ）调控的Ｃａ２＋－Ｍｇ２＋－ＡＴＰａｓｅ的活力影响。结果表明,在ＣａＭ和Ｃａ２＋－Ｍｇ２＋－ＡＴＰａｓｅ的体系中,一些Ｌｎ３＋（Ｌａ３＋、Ｇｄ３＋）对由ＣａＭ调节的Ｃａ２＋－Ｍｇ２＋－ＡＴＰａｓｅ的活力影响呈现双相效应,即Ｌｎ３＋在低浓度时,能提高激活Ｃａ２＋－Ｍｇ２＋－ＡＴＰａｓｅ的水解活力;在高浓度时,则抑制ＣａＭ调节Ｃａ２＋－Ｍｇ２＋－ＡＴＰａｓｅ活力的能力;少数Ｌｎ３＋（Ｓｍ３＋）仅表现出抑制效应。在无ＣａＭ的Ｃａ２＋－Ｍｇ２＋－ＡＴＰａｓｅ体系中,高浓度的Ｌｎ３＋抑制Ｃａ２＋－Ｍｇ２＋－ＡＴＰａｓｅ的基础活力。结合圆二色（ＣＤ）谱信息对Ｌｎ３＋和ＣａＭ相互作用的分子机制进行了初步的探讨。     

Ruthenium red inhibits the binding of calcium to calmodulin required for enzyme activation.

The Ca(2+)-calmodulin (CaM)-dependent activation of myosin light chain kinase is inhibited by ruthenium red competitively with respect to Ca2+, with a Ki value of 8.6 microM. The binding of Ca2+ to CaM is inhibited by micromolar concentrations of ruthenium red. In the absence of Ca2+, CaM has two binding sites for ruthenium red with the dissociation constants of 0.36 and 8.7 microM, respectively. Ca2+ antagonizes the binding of ruthenium red to the low-affinity site on CaM. Binding of ruthenium red to the high-affinity site is not affected by Ca2+. The low- and high-affinity sites for ruthenium red are shown to be located in the NH2-terminal half and the COOH-terminal half of CaM, respectively. Lower concentrations of ruthenium red are needed for enzyme inactivation than for the dissociation of enzyme-CaM-Sepharose complex, suggesting these events have different Ca2+ requirements. Moreover, ruthenium red inhibits Ca(2+)-induced contraction of depolarized vascular smooth muscle in a competitive manner with respect to Ca2+. These results suggest that ruthenium red may be a new type of CaM antagonist that inhibits the binding of Ca2+ to CaM and thereby inhibits Ca(2+)-CaM-dependent enzymes and smooth muscle contraction competitively with respect to Ca2+.     

Elucidation of conservative elements of calmodulin-dependent enzymes with the use of monoclonal antibodies

Monoclonal antibodies against human erythrocyte membrane Ca2+-ATPase were obtained. The binding of monoclonal antibodies to the enzyme resulted in a decrease in the enzyme sensitivity to calmodulin (CaM). The effects of monoclonal antibodies on other CaM-dependent enzymes, namely, on the phosphodiesterase of cAMP, phosphorylase kinase, and Ca2+-CaM-dependent protein kinase II (PK II), were studied. It was found that all four enzymes contain a common antigenic site. However, the inhibitory effect of antibodies was observed only with respect to Ca2+-ATPase and PK II. The kinetics of the binding of monoclonal antibodies and their inhibitory action were investigated. It was shown that the antigenic site is confined to the calmodulin-binding portion of Ca2+-ATPase and PK II.     

A new role for IQ motif proteins in regulating calmodulin function

IQ motifs are found in diverse families of calmodulin (CaM)-binding proteins. Some of these, like PEP-19 and RC3, are highly abundant in neuronal tissues, but being devoid of catalytic activity, their biological roles are not understood. We hypothesized that these IQ motif proteins might have unique effects on the Ca2+ binding properties of CaM, since they bind to CaM in the presence or absence of Ca2+. Here we show that PEP-19 accelerates by 40 to 50-fold both the slow association and dissociation of Ca2+ from the C-domain of free CaM, and we identify the sites of interaction between CaM and PEP-19 using NMR. Importantly, we demonstrate that PEP-19 can also increase the rate of dissociation of Ca2+ from CaM when bound to intact CaM-dependent protein kinase II. Thus, PEP-19, and presumably similar members of the IQ family of proteins, has the potential to alter the Ca2+-binding dynamics of free CaM and CaM that is bound to other target proteins. Since Ca2+ binding to the C-domain of CaM is the rate-limiting step for activation of CaM-dependent enzymes, the data reveal a new concept of importance in understanding the temporal dynamics of Ca2+-dependent cell signaling.     

Regulation of the dual specificity protein phosphatase, DsPTP1, through interactions with calmodulin

Reversible phosphorylation is a key mechanism for the control of intercellular events in eukaryotic cells. In animal cells, Ca2+/CaM-dependent protein phosphorylation and dephosphorylation are implicated in the regulation of a number of cellular processes. However, little is known on the functions of Ca2+/CaM-dependent protein kinases and phosphatases in Ca2+ signaling in plants. From an Arabidopsis expression library, we isolated cDNA encoding a dual specificity protein phosphatase 1, which is capable of hydrolyzing both phosphoserine/threonine and phosphotyrosine residues of the substrates. Using a gel overlay assay, we identified two Ca2+-dependent CaM binding domains (CaMBDI in the N terminus and CaMBDII in the C terminus). Specific binding of CaM to two CaMBD was confirmed by site-directed mutagenesis, a gel mobility shift assay, and a competition assay using a Ca2+/CaM-dependent enzyme. At increasing concentrations of CaM, the biochemical activity of dual specificity protein phosphatase 1 on the p-nitrophenyl phosphate (pNPP) substrate was increased, whereas activity on the phosphotyrosine of myelin basic protein (MBP) was inhibited. Our results collectively indicate that calmodulin differentially regulates the activity of protein phosphatase, dependent on the substrate. Based on these findings, we propose that the Ca2+ signaling pathway is mediated by CaM cross-talks with a protein phosphorylation signal pathway in plants via protein dephosphorylation.     

Cation- and peptide-binding properties of human calmodulin-like skin protein

Human CLSP, a new Ca(2+)-binding protein specifically expressed in differentiated keratinocytes, is a 15.9 kDa, four EF-hand containing protein with 52% sequence identity to calmodulin (CaM). The protein binds four Ca(2+) ions at two pairs of sites with [Ca(2+)](0.5) values of 1.2 and 150 microM, respectively. Mg(2+) at millimolar concentrations strongly decreases the affinity for Ca(2+) of the two high-affinity sites, but has no effect on the low-affinity sites. The protein can also bind two Mg(2+) ([Mg(2+)](0.5) = 57 microM) at the sites of high Ca(2+) affinity. Thus, as fast skeletal muscle troponin C (TnC), CLSP possesses two high-affinity Ca(2+)-Mg(2+) mixed sites and two low-affinity Ca(2+)-specific sites. Studies on the isolated recombinant N- (N-CLSP) and C-terminal half domains of CLSP (C-CLSP) revealed that, in contrast to the case of TNC, the high-affinity Ca(2+)-Mg(2+) mixed sites reside in the N-terminal half. The binding of cations modifies the intrinsic fluorescence of the two Tyr residues. Upon Ca(2+) binding, hydrophobicity is exposed at the protein surface that can be monitored with a fluorescent probe. The Ca(2+)-dependency of the two conformational changes is biphasic in the absence of Mg(2+), but monophasic in the presence of 2 mM Mg(2+), both corresponding closely to direct binding of Ca(2+) to CLSP. In the presence of Ca(2+), human CLSP forms a high-affinity 1:1 complex with melittin, a natural peptide considered to be a model for the interaction of CaM with its targets. In the complex, CLSP binds Ca(2+) with high affinity to all four binding sites. Isolated N- and C-CLSP show only a weak interaction with melittin, which is enhanced when both halves are simultaneously presented to the model peptide.     

Fe2+ and other divalent metal ions uncouple Ca2+ transport from (Ca2+-Mg2+)-ATPase in rat liver plasma membranes

The addition of nanomolar concentrations of free Fe2+, Mn2+, or Co2+ to rat liver plasma membranes resulted in an activation of ATP hydrolysis by these membranes which was not additive with the Ca2+-stimulated ATPase activity coupled to the Ca2+ pump. Detailed analysis showed that, if fact, (i) as for the stimulation of (Ca2+-Mg2+)-ATPase by Ca2+, activation of ATP hydrolysis by Fe2+, Mn3+, or Co2+ followed a cooperative mechanism involving two ions; (ii) two interacting sites for ATP were involved in the activation of both Fe2+- and Ca2+-stimulated ATPase activities; (iii) micromolar concentrations of magnesium caused the same dramatic inhibition of both activities; and (iv) the subcellular distribution of Fe2+-activated ATP hydrolysis activity corresponded to that of plasma membrane markers. This suggests that the (Ca2+-Mg2+)-ATPase might be stimulated not only by Ca2+, but also by Fe2+, Mn2+, or Co2+. However, interaction of (Ca2+-Mg2+)-ATPase with Fe2+, Mn2+, or Co2+ inhibited the Ca2+ pump activity. Furthermore, neither the formation of the phosphorylated intermediate of (Ca2+-Mg2+)-ATPase, nor ATP-dependent (59Fe) uptake could be detected in the presence of Fe2+ concentrations which stimulated ATP hydrolysis. We conclude that: (i) under the influence of certain metal ions, the Ca2+ pump in the liver plasma membrane may be switched to an uncoupled state which displays ATP hydrolysis activity, but does not insure ion transport; (ii) therefore the Ca2+ pump in liver plasma membranes specifically insures Ca2+ transport.     

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+).     

FRET-based in vivo Ca2+ imaging by a new calmodulin-GFP fusion molecule.

Intracellular Ca2+ acts as a second messenger that regulates numerous physiological cellular phenomena including development, differentiation and apoptosis. Cameleons, a class of fluorescent indicators for Ca2+ based on green fluorescent proteins (GFPs) and calmodulin (CaM), have proven to be a useful tool in measuring free Ca2+ concentrations in living cells. Traditional cameleons, however, have a small dynamic range of fluorescence resonance energy transfer (FRET), making subtle changes in Ca2+ concentrations difficult to detect and study in some cells and organelles. Using the NMR structure of CaM bound to the CaM binding peptide derived from CaM-dependent kinase kinase (CKKp), we have rationally designed a new cameleon that displays a two-fold increase in the FRET dynamic range within the physiologically significant range of cytoplasmic Ca2+ concentration of 0.05-1 microM.     

Auto-inhibition of Ca(2+)/calmodulin-dependent protein kinase II by its ATP-binding domain

Ca(2+)/calmodulin dependent protein kinase (CaMPK) II is a key enzyme in many physiological processes. The enzyme is inactive unless Ca(2+)/CaM binds to it. In this inactive form CaMPK-II does not bind ATP suggesting that the ATP-binding domain is involved in an intramolecular interaction. We show here that F12, a 12 amino acid long peptide fragment of the ATP-binding domain (CaMPK-II(23-34), GAFSVVRRCVKV) can inhibit the Ca(2+)/CaM-dependent activity (IC(50) of 3 microM) but has no effect on the Ca(2+)/CaM-independent activity of CaMPK-II. Kinetic analysis exhibited mixed inhibition with respect to autocamtide-2 and ATP. The inhibition by F12 showed specificity towards CaMPK-II, but also inhibited CaMPK-I (IC(50) = 12.5 microM), while CaMPK-IV (IC(50) = 85 microM) was inhibited poorly and cAMP-dependent protein kinase (PKA) was not inhibited. Substitution of phenylalanine at position 25 to alanine (A12), had little effect on the inhibition of different Ca(2+)/CaM-dependent protein kinases, suggesting that phenylalanine 25 does not play a crucial role in the interactions involving F12. Thus the molecular interactions involving the ATP-binding domain appears to play a role in the regulation of nonphosphorylated CaMPK-II activity.     

Separation and characteristics of inside-out and right side-out vesicles from a rat cardiac sarcolemma preparation

Purified cardiac sarcolemma (SL) vesicles are highly suitable to study various Ca2+-transport systems present in the SL. We describe in this paper the separation of the Inside-Out (IO) and Right side-Out (RO) oriented vesicle subpopulations from a purified rat heart SL preparation. The isolated subfractions were characterized with respect to the number of beta-adrenergic binding sites and the Ca2+-uptake and (Ca2+-Mg2+)-ATPase activities. It was found that the Ca2+-uptake and the (Ca2+-Mg2+)-ATPase activities reside in the IO fraction and are virtually absent in the RO fraction, confirming that the active Ca2+-uptake represents the outward directed sarcolemmal Ca2+-flux.