The ADP- and Mg2+-reactive calcium complex of the phosphoenzyme in skeletal sarcoplasmic reticulum Ca2+-ATPase

The effects of ATP on Ca²⁺ binding in the absence of added Mg²⁺ to the purified sarcoplasmic reticulum Ca²⁺-ATPase were studied at pH 7.0 and 0°C. ATP increased the number of Ca²⁺-binding sites of the enzyme from 2 to 3 mol per mol of phosphorylatable enzyme. The association constant for the ATP-induced Ca²⁺ binding was 4·10⁵ M^?1, which was not significantly different from that obtained in the absence of ATP. AdoP[CH₂]PP has little effect on the Ca²⁺-binding process. The amount of phosphoenzyme formed was equivalent to the level of ATP-induced Ca²⁺ binding. ADP decreased the level of ATP-induced Ca²⁺ binding and phosphoenzyme by the same amount. These results suggest that ATP-induced Ca²⁺ binding exists in the form of an ADP-reactive phosphoenzyme·Ca complex. In addition, the Ca²⁺ bound to the enzyme in the presence of ATP was released on the addition of 1 mM MgCl₂; after the release of Ca²⁺, the phosphoenzyme decayed. These observations suggest that Mg²⁺, added after the ATP-induced Ca²⁺-binding process, may replace the Ca²⁺ on the phosphoenzyme and initiate phosphoenzyme decomposition.     

The role of sialic acid in the nerve terminal for the release of transmitter

The role of sialic acid in the frequency of miniature endplate potentials (MEPPs) was examined using neuraminidase and gangliosides in the mouse diaphragm. Neuraminidase increased and decreased MEPP frequency in normal K⁺ and high K⁺ solution, respectively. The effects were dependent on the presence of Ca²⁺ in extracellular medium. Neuraminidase liberated sialic acid from and lowered Ca²⁺- binding capacity of synaptosomal membrane. Gangliosides treatment of the tissue partially restored the effects of neuraminidase on the frequency of MEPP and Ca²⁺-binding capacity. It is possible that sialic acid in the nerve endings provides a functional storage site which supply intracellular Ca²⁺ to cause a transmitter release.     

The interaction of Ca2+-binding proteins with the carbocyanine dye stains-all

The interaction of the carbocyanine dye Stains-all with the Ca²⁺-binding proteins calmodulin, troponin C, and parvalbumin has been monitored by means of absorption spectra and CD. In the absence of Ca²⁺, complexes with Stains-all of all three proteins exhibit at high dye: protein mole ratios an intense J absorption band at 600–650 nm, which is associated with a characteristic CD spectrum. In the cases of calmodulin and troponin C, the J-band is progressively lost as the dye: protein ratio decreases and is replaced by bands of the γ and β types at 450–550 nm, which likewise give rise to characteristic CD spectra. For parvalbumin, only the J-band is observed; its intensity is undiminished at the lowest dye: protein ratios examined. In the presence of excess Ca²⁺ the J-band is lost for all three proteins. For calmodulin and troponin C it is replaced by σ- and β-bands; in the case of parvalbumin the bound dye is released. A tentative model has been proposed to account for these observations.     

Equilibrium Dialysis Measurements of the Ca2+-Binding Properties of Recombinant Radish Vacuolar Ca2+-Binding Protein Expressed in Escherichia coli

Vacuoles of radish (Raphanus sativus) contained a Ca²⁺-binding protein (RVCaB) of 43 kDa. We investigated the Ca²⁺-binding properties of the protein. RVCaB was expressed in Escherichia coli and was purified from an extract by ion-exchange chromatography, nitrocellulose membrane filtration, and gel-filtration column chromatography. Ca²⁺-binding properties of the recombinant protein were examined by equilibrium dialysis with ⁴⁵Ca²⁺ and small dialysis buttons. The protein was estimated to bind 19Ca²⁺ ions per molecule with a K _d for Ca²⁺ of 3.4 mM. Ca²⁺ was bound to the protein even in the presence of high concentrations of Mg²⁺ or K⁺. The results suggested that the protein bound Ca²⁺ with high ion selectivity, high capacity, and low affinity.     

Calmodulin Mediates the Ca-Dependent Regulation of Cx44 Gap Junctions

We have shown previously that the Ca²⁺-dependent inhibition of lens epithelial cell-to-cell communication is mediated in part by the direct association of calmodulin (CaM) with connexin43 (Cx43), the major connexin in these cells. We now show that elevation of [Ca²⁺]_i in HeLa cells transfected with the lens fiber cell gap junction protein sheep Cx44 also results in the inhibition of cell-to-cell dye transfer. A peptide comprising the putative CaM binding domain (aa 129-150) of the intracellular loop region of this connexin exhibited a high affinity, stoichiometric interaction with Ca²⁺-CaM. NMR studies indicate that the binding of Cx44 peptide to CaM reflects a classical embracing mode of interaction. The interaction is an exothermic event that is both enthalpically and entropically driven in which electrostatic interactions play an important role. The binding of the Cx44 peptide to CaM increases the CaM intradomain cooperativity and enhances the Ca²⁺-binding affinities of the C-domain of CaM more than twofold by slowing the rate of Ca²⁺ release from the complex. Our data suggest a common mechanism by which the Ca²⁺-dependent inhibition of the α-class of gap junction proteins is mediated by the direct association of an intracellular loop region of these proteins with Ca²⁺-CaM.     

Inhibition of the Ca2+-and phospholipid-dependent protein kinase by a novel Mr 17,000 Ca2+-binding protein

A novel M_r 17,000 Ca²⁺-binding protein isolated from bovine brain was found to be a potent inhibitor of the Ca²⁺- and phospholipid-dependent protein kinase (protein kinase C), also isolated from bovine brain. Halfmaximal inhibition by this calciprotein of the initial rate of phosphorylation of histone III-S by protein kinase C occurred at a calciprotein concentration of 2.2 μM under standard conditions. Comparison of the effects of a number of Ca²⁺-binding proteins on protein kinase C activity indicated that the M_r 17,000 Ca²⁺-binding protein was the most potent inhibitor, followed by the intestinal Ca²⁺-binding protein and calcineurin. Calmodulin, troponin C, S-100 protein and a M_r 21,000 Ca²⁺-binding protein of bovine brain were relatively weak inhibitors of protein kinase C. The inhibitory effect of the M_r 17,000 Ca²⁺-binding protein was apparently not due to its interaction with phospholipid or the basic protein substrate and therefore appears to be due to a direct effect on the protein kinase C. These observations suggest that the novel M_r 17,000 Ca²⁺-binding protein, and possibly other Ca²⁺-binding proteins, may play a physiological role in regulating the activity of protein kinase C.     

Ca2+, pH and the regulation of cardiac myofilament force and ATPase activity

When the (pH_i) surrounding myofilaments of striated muscle is reduced there is an inhibition of both the actin-myosin reaction as well as the Ca²⁺-sensitivity of the myofilaments. Although the mechanism for the effect of acidic pH on Ca²⁺-sensitivity has been controversial, we have evidence for the hypothesis that acidic pH reduces the affinity of troponin C (TNC) for Ca²⁺. This effect of acidic pH depends not only on a direct effect of protons on Ca²⁺-binding to TNC, but also upon neighboring thin filament proteins, especially TNI, the inhibitory component of the TN complex. Using flourescent probes that report Ca²⁺-binding to the regulatory sites of skeletal and cardiac TNC, we have shown, for example, that acidic pH directly decreases the Ca²⁺-affinity of TNC, but only by a relatively small amount. However, with TNC in whole TN or in the TNI-TNC complex, there is about a 2-fold enhancement of the effects of acidic pH on Ca²⁺-binding to TNC. Acidic pH decreases the affinity of skeletal TNI for skeletal TNC, and also influences the micro-environment of a probe postioned at Cys-133 of TNI, a region of interaction with TNC. Other evidence that the effects of acidic pH on Ca²⁺-TNC activation of myofilaments are influenced by TNI comes from studies with developing hearts. In contrast, to the case with the adult preparations, Ca²⁺-activation of detergent extracted fibers prepared from dog or rat hearts in the peri-natal period are weakly affected by a drop in pH from 7.0 to 6.5. This difference in the effect of acidic (pH_i) appears to be due to a difference in the isoform population of TNI, and not to differences in isotype population or amount of TNC.     

The estimation of free calcium within synaptosomes and mitochondria with fura-2; comparison to quin-2

The utility of the acetoxymethyl esters of two tetracarboxylic acids, fura-2 and quin-2, in the determination of ionic calcium levels within synaptosomes and mitochondria was compared. Synaptosomes and isolated mitochondria both accumulated the esters but mitochondria had a much more limited capacity to hydrolyze them. Dye-loaded synaptosomes maintain their external membrane potential of magnitude similar to values for unloaded controls and do not accumulate radioactive Ca²⁺ in excess with time. Both fluorescent compounds yielded similar values (about 300–400 nM) for free intrasynaptosomal calcium [Ca²⁺]_i. Mitochondrial Ca²⁺ could be measured only with fura-2. Isolated mitochondria contained 0.9–1 μM free Ca²⁺ in a similar extrasynaptosomal medium. Fura-2 tended to overestimate [Ca²⁺]_i while quin-2 tended to underestimate it due to chelation of these dyes with intrasynaptosomal trace elements. Fura-2 requiring the use of two excitation wavelengths was significantly superior to the single wavelength method using quin-2. Advantages included reduced danger of erroneous readings due to (i) synaptosomal sedimentation, (ii) photobleaching of the dye, (iii) underestimation of intrasynaptosomal calcium during correction for dye leakage by manganese entry into synaptosomes. Fura-2 interfered less with synaptosomal Ca²⁺ transients than quin-2, probably due to lower intrasynaptosomal concentration of dye needed. Both unstimulated and K⁺-stimulated ⁴⁵Ca²⁺ uptake were increased in quin-2-loaded synaptosomes but only K⁺-stimulated uptake in fura-2 loaded ones. This series of advantages makes fura-2 of superior utility in the determination of free intrasynaptosomal calcium.     

Disulfiram lowers Ca2+, Mg2+-ATPase activity of rat brain synaptosomes

The chronic administration of disulfiram (DS) to rats resulted in significant decrease of synaptosomal Ca²⁺, Mg²⁺-ATPase activity. In vitro studies indicated that DS (ID₅₀=20 M) produced a dose-dependent inhibition of Ca²⁺, Mg²⁺-ATPase. However, diethyldithio-carbamate, a metabolite of DS, failed to modify Ca²⁺, Mg²⁺-ATPase activity, implying that the decrease in ATPase activity in DS administered rats was due to the effect of parent compound. The DS-mediated inhibition (48%) of ATPase activity was comparable with a similar degree of inhibition (49%) achieved by treating the synaptosomal membranes with N-ethylmaleimide (ID₅₀=20 M) in vitro. Furthermore, the inhibition by DS was neither altered by washing the membranes with EGTA nor reversed by treatment with sulfhydryl reagents such as GSH or dithiothreitol. About 74% and 68% decrease of synaptosomal Ca²⁺, Mg²⁺-ATPase specific activity was observed when treated with DS (30 M) and EGTA (100 M) respectively. The remaining 25–30% of total activity is suggested to be of Mg²⁺-dependent ATPase activity. This indicates that both these drugs may act on a common target, calmodulin component that represents 70–75% of total Ca²⁺, Mg²⁺-ATPase activity. Therefore, DS-mediated modulation of synaptosomal Ca²⁺, Mg²⁺-ATPase activity could affect its function of maintaining intracellular Ca²⁺ concentration. This could contribute to the deleterious effects on CNS.     

The binding of arsenazo III to cell components

The Ca²⁺ indicator, arsenazo III, binds to subcellular fractions of rabbit skeletal muscle with sufficient affinity that in living muscle containing 1–2 mM arsenazo III, the estimated free arsenazo III concentration is only 50–200 μM; 80–90% of the bound arsenazo III is associated with soluble proteins.The binding of arsenazo III to soluble proteins decreases the optical response of the dye to Ca²⁺; this is due to a decrease in the affinity of the protein-bound dye for Ca²⁺. Approximately half of the bound arsenazo III is released from the particulate fraction and soluble proteins upon addition of 5 mM Ca²⁺, suggesting that the Ca-arsenazo complex has lower affinity for the protein binding sites than the free dye.The Ca²⁺ binding to the soluble protein fraction of rabbit skeletal muscle is attributable largely to its parvalbumin content.     

Isolation of sarcoplasmic reticulum by zonal centrifugation and purification of Ca2+-pump and Ca2+-binding proteins

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.     

Oligomerization of the Polycystin-2 C-terminal Tail and Effects on Its Ca2+-binding Properties

Polycystin-2 (PC2) belongs to the transient receptor potential (TRP) family and forms a Ca²⁺-regulated channel. The C-terminal cytoplasmic tail of human PC2 (HPC2 Cterm) is important for PC2 channel assembly and regulation. In this study, we characterized the oligomeric states and Ca²⁺-binding profiles in the C-terminal tail using biophysical approaches. Specifically, we determined that HPC2 Cterm forms a trimer in solution with and without Ca²⁺ bound, although TRP channels are believed to be tetramers. We found that there is only one Ca²⁺-binding site in the HPC2 Cterm, located within its EF-hand domain. However, the Ca²⁺ binding affinity of the HPC2 Cterm trimer is greatly enhanced relative to the intrinsic binding affinity of the isolated EF-hand domain. We also employed the sea urchin PC2 (SUPC2) as a model for biophysical and structural characterization. The sea urchin C-terminal construct (SUPC2 Ccore) also forms trimers in solution, independent of Ca²⁺ binding. In contrast to the human PC2, the SUPC2 Ccore contains two cooperative Ca²⁺-binding sites within its EF-hand domain. Consequently, trimerization does not further improve the affinity of Ca²⁺ binding in the SUPC2 Ccore relative to the isolated EF-hand domain. Using NMR, we localized the Ca²⁺-binding sites in the SUPC2 Ccore and characterized the conformational changes in its EF-hand domain due to trimer formation. Our study provides a structural basis for understanding the Ca²⁺-dependent regulation of the PC2 channel by its cytosolic C-terminal domain. The improved methodology also serves as a good strategy to characterize other Ca²⁺-binding proteins.     

Slow transition of phosphoenzyme from ADP-sensitive to ADP-insensitive forms in solubilized Ca2+, Mg2+-ATPase of sarcoplasmic reticulum: evidence for retarded dissociation of Ca2+ from the phosphoenzyme.

Solubilized Ca²⁺, Mg²⁺-ATPase of sarcoplasmic reticulum was phosphorylated with ATP without added MgCl₂. The phosphoenzyme formed was ADP-sensitive. Ca²⁺ in the medium was chelated after phosphorylation. This induced a slow transition of the phosphoenzyme from ADP-sensitive to ADP-insensitive forms. The ADP-sensitivity was restored by subsequent addition of CaCl₂. These results showed that the transition was caused by dissociation of Ca²⁺ bound to the phosphoenzyme. Further observations indicated that, when Ca²⁺ in the medium was chelated, Ca²⁺ bound to the phosphoenzyme was dissociated much more slowly than Ca²⁺ bound to the dephosphoenzyme. This suggests a possible formation of the occluded form of the Ca²⁺-binding site in the phosphoenzyme.     

Relationship between synaptosomal uptake and release of [14C]gaba, [14C]diaminobutyric acid and [14C]beta-alanine.

[¹⁴C]GABA is taken up by rat brain synaptosomes via a high affinity, Na⁺-dependent process. Subsequent addition of depolarizing levels of potassium (56.2 MM) or veratridine (100 μM) stimulates the release of synaptosomal [¹⁴C]GABA by a process which is sensitive to the external concentration of divalent cations such as Ca²⁺, Mg²⁺, and Mn²⁺. However, the relatively smaller amount of [¹⁴C]GABA taken up by synaptosomes in the absence of Na⁺ is not released from synaptosomes by Ca²⁺ -dependent, K ⁺-stimulation. [¹⁴C]DABA, a competitive inhibitor of synaptosomal uptake of GABA (Iversen & Johnson , 1971) is also taken up by synaptosomal fractions via a Na ⁺ -dependent process; and is subsequently released by Ca²⁺ -dependent, K⁺-stimulation. On the other hand, [¹⁴C]β-alanine, a purported blocker of glial uptake systems for GABA (Schon & Kelly , 1974) is a poor competitor of GABA uptake into synaptosomes. Comparatively small amounts of [¹⁴C] β-alanine are taken up by synaptosomes and no significant amount is released by Ca²⁺ -dependent, K⁺-stimulation. These data suggest that entry of [¹⁴C]GABA into a releasable pool requires external Na⁺ ions and maximal evoked release of [¹⁴C]GABA from the synaptosomal pool requires external Ca²⁺ ions. The GABA analogue, DABA, is apparently successful in entering the same or similar synaptosomal pool. The GABA analogue, β-alanine, is not. None of the compounds or conditions studied were found to simultaneously affect both uptake and release processes. Compounds which stimulated release (veratridine) or inhibited release (magnesium) were found to have minimal effect on synaptosomal uptake. Likewise compounds (DABA) or conditions (Na⁺-free medium) which inhibited uptake, had little effect on release.     

Ca-dependent binding of calcium-binding protein 1 to presynaptic group III metabotropic glutamate receptors and blockage by phosphorylation of the receptors

Presynaptic group III metabotropic glutamate receptors (mGluRs) and Ca²⁺ channels are the main neuronal activity-dependent regulators of synaptic vesicle release, and they use common molecules in their signaling cascades. Among these, calmodulin (CaM) and the related EF-hand Ca²⁺-binding proteins are of particular importance as sensors of presynaptic Ca²⁺, and a multiple of them are indeed utilized in the signaling of Ca²⁺ channels. However, despite its conserved structure, CaM is the only known EF-hand Ca²⁺-binding protein for signaling by presynaptic group III mGluRs. Because the mGluRs and Ca²⁺ channels reciprocally regulate each other and functionally converge on the regulation of synaptic vesicle release, the mGluRs would be expected to utilize more EF-hand Ca²⁺-binding proteins in their signaling. Here I show that calcium-binding protein 1 (CaBP1) bound to presynaptic group III mGluRs competitively with CaM in a Ca²⁺-dependent manner and that this binding was blocked by protein kinase C (PKC)-mediated phosphorylation of these receptors. As previously shown for CaM, these results indicate the importance of CaBP1 in signal cross talk at presynaptic group III mGluRs, which includes many molecules such as cAMP, Ca²⁺, PKC, G protein, and Munc18-1. However, because the functional diversity of EF-hand calcium-binding proteins is extraordinary, as exemplified by the regulation of Ca²⁺ channels, CaBP1 would provide a distinct way by which presynaptic group III mGluRs fine-tune synaptic transmission.     

Parallel measurements of bound calcium and force in glycerinated rabbit psoas muscle fibers

A simple double-isotope procedure has been developed for making simultaneous measurements of bound Ca²⁺ and relative force in glycerinated rabbit psoas bundles containing two fibers. With this preparation it is possible to study Ca²⁺-troponin interactions coincident with MgATP-induced force development. Over the free [Ca²⁺] range 6 · 10^?8–1.2 · 10^?5 M the bound Ca²⁺ varied from 0.25 to 1.65 μmol/g protein. The free [Ca²⁺] at half-maximal Ca²⁺ saturation was 2 · 10^?7 M while that a half-maximal force was 5 · 10^?7 M. Half-maximal Ca²⁺ saturation was associated with 20% maximal force. The force-[Ca²⁺] saturation curve showed a steep rise in slope at greater than half saturation. The observed relationship was consistent with a model in which multiple occupancy of troponin Ca²⁺-binding sites is essential for initiation of cross-bridge cycling.     

Opiates inhibit calmodulin activation of a high-affinity Ca2+-stimulated Mg2+-dependent ATPase in synaptic membranes

A high affinity Ca²⁺/Mg²⁺ ATPase has been identified and localized in synaptic membrane subfractions. This enzyme is stimulated by low concentrations of Ca²⁺ (1 M) believed to approximate the range of Ca²⁺ in the synaptosomal cytosol (0.1 to 5.0 M). The opiate agonist levorphanol, in a concentration-dependent fashion, inhibited Ca²⁺-stimulated ATP hydrolysis in lysed synaptic membranes. This inhibition was reversed by naloxone, while dextrorphan, the inactive opiate isomer, was without effect. Inhibition by levorphanol was most pronounced in a subfraction of synaptic membranes (SPM-1). The inhibition of Ca²⁺-stimulated ATP hydrolysis was characterized by a reduction inV _max for Ca²⁺. Levorphanol pretreatment reduced the Hill coefficient (H_N) of 1.5 to 0.7, suggesting cooperative interaction between the opiate receptor and the enzyme protein. Levorphanol, but not dextrorphan, also inhibited (28%) ATP-dependent Ca²⁺ uptake by synaptic membranes. Opiate ligand stereoisomers were tested for their effects on calmodulin stimulating of high affinity Ca²⁺/Mg²⁺ ATPase in synaptic membranes. Levorphanol (10 M), but not the inactive stereoisomer (+)dextrorphan, significantly inhibited (35%) the calmodulin-activated Ca²⁺-dependent ATP hydrolysis activity in a preparation of lysed synaptic membranes. Both Ca²⁺-dependent and calmodulin-dependent stimulation of the enzyme in the presence of optimal concentrations of the other co-substrate were inhibited by levorphanol (35–40%) but not dextrorphan. Inhibition of ATP hydrolysis was characterized by a reduction inV _max for both Ca²⁺ and calmodulin stimulation of the enzyme. Calmodulin stimulation of enzyme activity was most pronounced in SPM-1, the membrane fraction which also exhibits the maximal opiate inhibition (40%) of the Ca²⁺-ATPase. The results demonstrate that opiate receptor activation inhibits a high affinity Ca²⁺/Mg²⁺ ATPase in synaptic plasma membranes in a stereospecific fashion. The inhibition of the enzyme may occur by a mechanism involving both Ca²⁺ and calmodulin. Inhibition of calmodulin activation may contribute to the mechanism by which opiate ligands disrupt synaptosomal Ca²⁺ buffering mechanisms. Changes in the cytosolic distribution of synaptosomal Ca²⁺ following inhibition of Ca²⁺/Mg²⁺ ATPase may underlie some of the pharmacological effects of opiate drugs.     

Inhibition by N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide,a calmodulin inhibitor,of dopamine release from rat brain synaptosomes

The release of preloaded [³H]dopamine by the synaptosomal fraction prepared from rat forebrain was examined in the presence and absence of N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a calmodulin inhibitor. The release induced by high K⁺ was blocked by W-7 in a concentration-dependent manner after the pretreatment with and in the presence of the inhibitor. The inhibition by W-7 may specifically involve calmodulin, because little effects were seen with N-(6-aminohexyl)-naphthalenesulfonamide, an analog of W-7 with only a low affinity for calmodulin. W-7 may not affect the voltage-dependent Ca²⁺ channel of synaptosomal plasmalemma, since the inhibitor produced no change in the synaptosomal ⁴⁵Ca²⁺ uptake induced by high K⁺ depolarization. Thus, calmodulin may play a role in transmitter release and may function at the step(s) after the increase of free Ca²⁺ concentration in the cytosol of the nerve terminal. W-7 affected only to a small extent [³H]dopamine release in the presence of A23187 plus Ca²⁺.     

A rapid induction by elicitors of the mRNA encoding CCD-1, a 14 kDa Ca2+-binding protein in wheat cultured cells

Intracellular Ca²⁺ has been implicated in the signal transduction processes during the development of the plant defense system against fungal pathogens. From wheat cultured cells that had been treated with the elicitor derived from Typhula ishikariensis, the ccd-1 gene encoding a 14 kDa Ca²⁺-binding protein with an acidic amphiphilic feature was isolated. The ccd-1-encoded protein (CCD-1) shares homology to the C-terminal half domain of centrin, a Ca²⁺-binding protein conserved in eukaryotes. Unlike typical eukaryotic centrins, CCD-1 contains only one Ca²⁺-binding loop, which corresponds to the one in the fourth EF-hand from the N-terminus of centrin. The recombinant CCD protein expressed in Escherichia coli bound to a phenyl-Sepharose column in the presence of Ca²⁺ and was eluted out by EGTA. It also showed a Ca²⁺-dependent electrophoretic mobility shift on the non-denaturing polyacrylamide gel. The ccd-1 mRNA expression was rapidly induced by treatment with fungal and chitosan oligosaccharide elicitors, implying that it might have a role in transducing Ca²⁺ signals provoked by the elicitors. The expression of the ccd-1 mRNA was induced by treatment with A23187, and the induction was suppressed by La³⁺ or 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid (BAPTA). This study suggests the involvement of intracellular Ca²⁺ in the elicitor-induced mRNA expression of a novel class of Ca²⁺-binding proteins conserved in higher plants.     

Effects of calmodulin on the (Ca2+ + Mg2+)ATPase partially purified from erythrocyte membranes.

The stimulation of the (Ca²⁺ + Mg²⁺)ATPase of erythrocyte ghosts by calmodulin was observed not only in intact ghosts, but also in the solubilized (Triton X-100) and partially purified, reconstituted (phosphatidylserine liposomes) forms. Since the solubilized form of the enzyme migrated on Sepharose 6B at a position corresponding to a molecular weight of about 150,000, these results show that calmodulin stimulates by direct interaction with the ATPase complex. Additionally, the effects of calmodulin on erythrocyte ghosts prepared by the Dodge-EDTA method (hypotonic ghosts) and by the method of Ronner et al. (involving lysis followed by an isotonic wash repeated several times) were compared (P. Ronner, P. Gazzotti, and E. Carafoli, 1977, Arch. Biochem. Biophys. 179, 578–583). The (Ca²⁺ + Mg²⁺)ATPase of the hypotonic ghosts was low and was stimulated by added calmodulin while that of the isotonic ghosts was high and changed only slightly upon calmodulin addition; this difference in response to calmodulin persisted in the solubilized and reconstituted forms. Hypotonic ghosts bound ¹²⁵I-labeled calmodulin, while isotonic ghosts did not. This comparison of two types of ghosts showed that isotonic ghosts possess an intact calmodulin-(Ca²⁺ + Mg²⁺)ATPase complex, and that the calmodulin remained with the ATPase during solubilization and reconstitution. The isotonic preparation is a particularly useful method of preparing ghosts with an intact calmodulin-ATPase complex, since it requires no special equipment and produces an enzyme activity which is stable to freezing.