Ca(2+)- and H(+)-dependent conformational changes of calbindin D(28k)

Calbindin D(28k) is a member of a large family of intracellular Ca(2+) binding proteins characterized by EF-hand structural motifs. Some of these proteins are classified as Ca(2+)-sensor proteins, since they are involved in transducing intracellular Ca(2+) signals by exposing a hydrophobic patch on the protein surface in response to Ca(2+) binding. The hydrophobic patch serves as an interaction site for target enzymes. Other members of this group are classified as Ca(2+)-buffering proteins, because they remain closed after Ca(2+) binding and participate in Ca(2+) buffering and transport functions. ANS (8-anilinonaphthalene-1-sulfonic acid) binding and affinity chromatography on a hydrophobic column suggested that both the Ca(2+)-free and Ca(2+)-loaded form of calbindin D(28k) have exposed hydrophobic surfaces. Since exposure of hydrophobic surface is unfavorable in the aqueous intracellular milieu, calbindin D(28k) most likely interacts with other cellular components in vivo. A Ca(2+)-induced conformational change was readily detected by several optical spectroscopic methods. Thus, calbindin D(28k) shares some of the properties of Ca(2+)-sensor proteins. However, the Ca(2+)-induced change in exposed hydrophobic surface was considerably less pronounced than that in calmodulin. The data also shows that calbindin D(28k) undergoes a rapid and reversible conformational change in response to a H(+) concentration increase within the physiological pH range. The pH-dependent conformational change was shown to reside mainly in EF-hands 1-3. Urea-induced unfolding of the protein at pH 6, 7, and 8 showed that the stability of calbindin D(28k) was increased in response to H(+) in the range examined. The results suggest that calbindin D(28k) may interact with targets in a Ca(2+)- and H(+)-dependent manner.     

Calcium-modulated S100 protein-phospholipid interactions. An NMR study of calbindin D9k and DPC

The cellular functions of several S100 proteins involve specific interactions with phospholipids and the cell membrane. The interactions between calbindin D(9k) (S100D) and the detergent dodecyl phosphocholine (DPC) were studied using NMR spectroscopy. In the absence of Ca(2+), the protein associates with DPC micelles. The micelle-associated state has intact helical secondary structures but no apparent tertiary fold. At neutral pH, Ca(2+)-loaded calbindin D(9k) does not associate with DPC micelles. However, a specific interaction is observed with individual DPC molecules at a site close to the linker between the two EF-hands. Binding to this site occurs only when Ca(2+) is bound to the protein. A reduction in pH in the absence of Ca(2+) increases the stability of the micelle-associated state. This along with the corresponding reduction in Ca(2+) affinity causes a transition to the micelle-associated state also in the presence of Ca(2+) when the pH is lowered. Site-specific analysis of the data indicates that calbindin D(9k) has a core of three tightly packed helices (A, B, and D), with a dynamic fourth helix (C) more loosely associated. Evidence is presented that the Ca(2+)-binding characteristics of the two EF-hands are distinctly different in a micelle environment. The role of calbindin D(9k) in the cell is discussed, along with the broader implications for the function of the S100 protein family.     

Calbindin D28k exhibits properties characteristic of a Ca2+ sensor

Calbindin D(28k) is a member of the calmodulin superfamily of Ca(2+)-binding proteins and contains six EF-hands. The protein is generally believed to function as a Ca(2+) buffer, but the studies presented in this work indicate that it may also act as a Ca(2+) sensor. The results show that Mg(2+) binds to the same sites as Ca(2+) with an association constant of approximately 1.4.10(3) m(-1) in 0.15 m KCl. The four high affinity sites in calbindin D(28k) bind Ca(2+) in a non-sequential, parallel manner. In the presence of physiological concentrations of Mg(2+), the Ca(2+) affinity is reduced by a factor of 2, and the cooperativity, which otherwise is modest, increases. Based on the binding constants determined in the presence of physiological salt concentrations, we estimate that at the Ca(2+) concentration in a resting cell calbindin D(28k) is saturated to 40-75% with Mg(2+) but to less than 9% with Ca(2+). In contrast, the protein is expected to be nearly fully saturated with Ca(2+) at the Ca(2+) level of an activated cell. A substantial conformational change is observed upon Ca(2+) binding, but only minor structural changes take place upon Mg(2+) binding. This suggests that calbindin D(28k) undergoes Ca(2+)-induced structural changes upon Ca(2+) activation of a cell. Thus, calbindin D(28k) displays several properties that would be expected for a protein involved in Ca(2+)-induced signal transmission and hence may function not only as a Ca(2+) buffer but also as a Ca(2+) sensor. Digestion patterns resulting from limited proteolysis of the protein suggest that the loop of EF-hand 2, a variant site that does not bind Ca(2+), becomes exposed upon Ca(2+) binding.     

Kinetic mechanism of the Zn-dependent aryl-phosphatase activity of myo-inositol-1-phosphatase

Myo-inositol-1-phosphatase (EC 3.1.3.25) is able to hydrolyze myo-inositol-1-phosphate in the presence of Mg(2+) ions at neutral pH, and also p-nitrophenyl phosphate in the presence of Zn(2+)-ions at acidic pH. This enzyme plays a role in phosphatidylinositol cell signalling and is a putative target of lithium therapy in manic depression. We elucidate here the kinetic mechanism of the Zn-dependent activity of myo-inositol-1-phosphatase. As part of this analysis it was necessary to determine the basicity constants of p-nitrophenyl phosphate and the stability constant of its metal-complex in the presence of zinc chloride. We find that the Zn-dependent reaction may be described either by a rapid-equilibrium random mechanism or an ordered steady-state mechanism in which the substrate binds to the free enzyme prior to the metal ion. In both models the Zn-substrate complex acts as a high affinity inhibitor, yielding a dead-end species through its binding to the enzyme-Zn-substrate in rapid-equilibrium or to the enzyme-phosphate complexes in a steady-state model. Phosphate is a competitive inhibitor of the enzyme with respect to the substrate and an uncompetitive inhibitor with respect to zinc ions.     

NMDA-induced 45Ca release in the dentate gyrus of newborn rats: in vivo microdialysis study

This in vivo study, aimed at detecting the N-methyl-D-aspartate (NMDA) evoked Ca(2+)-induced Ca(2+) release from intracellular stores in the neonatal rat brain, demonstrates that the application of 5 mM N-methyl-D-aspartate via a microdialysis probe for 20 min to the dentate gyrus (DG) of halotane-anesthetized 7 day-old (postnatal day 7, PND 7) rats induces a prolonged decrease in Ca(2+) concentration in an initially calcium-free dialysis medium, indicative of a drop in the extracellular concentration of Ca(2+) and Ca(2+) influx to neurons. In parallel experiments, a huge NMDA-evoked release of 45Ca from the pre-labeled endogenous Ca(2+) pool was observed and interpreted as the expression of intracellular Ca(2+) release. Dantrolene (100 microM) significantly inhibited the NMDA-induced 45Ca release, whereas 250 microM ryanodine exerted an unspecific biphasic effect. Autoradiographic and immunocytochemical detection of ryanodine receptors and calbindin D(28K), respectively, in the hippocampal region of PND 7 rats displayed a pronounced expression of [3H]ryanodine binding sites in the DG, but only a slight immunoreactivity of calbindin D(28K). Plastic changes in neurons or excitotoxic neuronal damage induced by the activation of NMDA receptors are mediated by Ca(2+) signals, resulting from an influx of extracellular Ca(2+), and also in some neurons, from the release of intracellular Ca(2+). Our previous in vivo microdialysis experiments visualized NMDA-evoked 45Ca release in the adult rat dentate gyrus, attributable to Ca(2+)-induced Ca(2+) release from the ryanodine-sensitive pool. An additional role of calbindin in the mechanism of this phenomenon has been suggested. This aspect has not been studied in vivo in newborn rats. Our present results indicate that the release of 45Ca from the prelabeled intracellular, dantrolene-sensitive Ca(2+) pool in the DG neurons of immature rats, most probably representing a phenomenon of Ca(2+)-induced Ca(2+) release, significantly participates in the generation of NMDA receptor-mediated intracellular Ca(2+) signals, whereas the role of calbindin D(28K) in the mechanism of 45Ca release is negligible.     

Calbindin D(9k): a protein optimized for calcium binding at neutral pH.

The binding of calcium ions by EF-hand proteins depends strongly on the electrostatic interactions between Ca(2+) ions and negatively charged residues of these proteins. We have investigated the pH dependence of the binding of Ca(2+) ions by calbindin D(9k). This protein offers a unique possibility for interpretation of such data since the pK(a) values of all ionizable groups are known. The binding is independent of pH between 7 and 9, where maximum calcium affinity is observed. An abrupt decrease in the binding affinity is observed at pH values below 7. This decrease is due to protonation of acidic groups, leading to modification of protein charges. The pH dependence of the product of the two macroscopic Ca(2+)-binding constants can be formally described by the involvement of two acidic groups with pK(a) = 6.6. Monte Carlo calculations show that the reduction of Ca(2+) binding is strictly determined by variable electrostatic interactions due to pH-dependent changes not only in the binding sites, but also of the overall charge of the protein.     

Plasma Membrane Ca-ATPase of Radish Seedlings : I. Biochemical Characterization Using ITP as a Substrate

In this work, we exploited the capability of the plasma membrane Ca-ATPase to utilize ITP as a substrate to study its characteristics in plasma membrane vesicles purified from radish (Raphanus sativus L.) seedlings. The majority of the ITPase activity of plasma membrane was Ca(2+)-dependent. The Ca(2+)-dependent ITPase activity was Mg(2+)-dependent and was stimulated by the calcium ionophore A23187. It was inhibited by erythrosin B (concentration giving 50% inhibition, 50 nanomolar) and by vanadate (concentration giving 50% inhibition, 3 micromolar) and displayed a broad pH optimum around pH 7.2 to 7.5. Both the hydrolytic and the transport activity of the plasma membrane Ca-ATPase were half-saturated by Ca(2+) in the micromolar concentration range. No major effect of EGTA on the saturation kinetics of the enzyme was observed. The affinity of the plasma membrane Ca-ATPase for Ca(2+) was about fourfold higher at pH 7.5 than at pH 6.9. The Ca(2+)-dependent ITPase activity was stimulated about twofold by polyoxyethylene 20 cetyl ether, although it was inhibited by Triton X-100 and by lysolecithin.     

Mechanisms underlying the hemolytic and ichthyotoxic activities of maitotoxin.

Maitotoxin (MTX), a putative Ca(2+) channel activator produced by the dinoflagellate Gambierdiscus toxicus showed extremely potent hemolytic and ichthyotoxic activities. Hemolysis of 1% mouse blood cell suspension in saline occurred at 15 nM of MTX. The activity was enhanced six-fold in the presence of 10 microM of Ca(2+) and completely blocked by EDTA2Na, indicating its dependency on external Ca(2+). The MTX-induced hemolysis was little affected by L-type Ca(2+) channel blockers (diltiazem, nifedipine, verapamil) but was strongly inhibited by calmodulin blockers (prenylamine and chlorpromazine) or a phospholipase A2 inhibitor (quinacrine). MTX was mimicked by a calcium ionophore, calcimycin. Based on these results, a series of cellular events triggered by MTX were presumed to occur in the following sequence: increased Ca(2+) entry in cells, activation of calmodulin, promotion of phospholipase A2 activity, and finally destruction of cell membrane resulting from hydrolysis of membrane lipids. The sensitivity of blood cells to MTX varied significantly, dependent on the animal sources. Nucleated blood cells of carps and chickens were 100 times more resistant than those of mammals. LC(50) of MTX to freshwater fish Tanichthys albonubes in Ca(2+) free media (pH 8) was 5 nM but was markedly lowered to 3 pM by raising pH to 8 and increasing Ca(2+) concentration to 2 mM. In a marine environment MTX was 2000 times more toxic to fish than 42-di-hydrobrevetoxin-B (PbTx-3), one of the best known ichthyotoxins of red-tide origins.     

Deamidation and disulfide bridge formation in human calbindin D28k with effects on calcium binding

Calbindin D(28k) (calbindin) is a cytoplasmic protein expressed in the central nervous system, which is implied in Ca(2+) homeostasis and enzyme regulation. A combination of biochemical methods and mass spectrometry has been used to identify post-translational modifications of human calbindin. The protein was studied at 37 degrees C or 50 degrees C in the presence or absence of Ca(2+). One deamidation site was identified at position 203 (Asn) under all conditions. Kinetic experiments show that deamidation of Asn 203 occurs at a rate of 0.023 h(-1) at 50 degrees C for Ca(2+)-free calbindin. Deamidation is slower for the Ca(2+)-saturated protein. The deamidation process leads to two Asp iso-forms, regular Asp and iso-Asp. The form with regular Asp 203 binds four Ca(2+) ions with high affinity and positive cooperativity, i.e., in a very similar manner to non-deamidated protein. The form with beta-aspartic acid (or iso-Asp 203) has reduced affinity for two or three sites leading to sequential Ca(2+) binding, i.e., the Ca(2+)-binding properties are significantly perturbed. The status of the cysteine residues was also assessed. Under nonreducing conditions, cysteines 94 and 100 were found both in reduced and oxidized form, in the latter case in an intramolecular disulfide bond. In contrast, cysteines 187, 219, and 257 were not involved in any disulfide bonds. Both the reduced and oxidized forms of the protein bind four Ca(2+) ions with high affinity in a parallel manner and with positive cooperativity.     

Pore formation and uncoupling initiate a Ca2+-independent degradation of mitochondrial phospholipids

Mitochondria contain a type IIA secretory phospholipase A(2) that has been thought to hydrolyze phospholipids following Ca(2+) accumulation and induction of the permeability transition. These enzymes normally require millimolar Ca(2+) for optimal activity; however, no dependence of the mitochondrial activity on Ca(2+) can be demonstrated upon equilibrating the matrix space with extramitochondrial Ca(2+) buffers. Ca(2+)-independent activity is seen following protonophore-mediated uncoupling, when uncoupling arises through alamethicin-mediated pore formation, or upon opening the permeability transition pore. Under the latter conditions, activity continues in the presence of excess EGTA but is somewhat enhanced by exogenous Ca(2+). The Ca(2+)-independent activity is best seen in media of high ionic strength and displays a broad pH optimum located between pH 8 and pH 8.5. It is strongly inhibited by bromoenol lactone but not by arachidonyl trifluoromethyl ketone, dithiothreitol, and other inhibitors of particular phospholipase A(2) classes. Immunoanalysis of mitochondria and mitochondrial subfractions shows that a membrane-bound protein is present that is recognized by antibody against an authentic iPLA(2) that was first found in P388D(1) cells. It is concluded that mitochondria contain a distinct Ca(2+)-independent phospholipase A(2) that is regulated by bioenergetic parameters. It is proposed that this enzyme, rather than the Ca(2+)-dependent type IIA phospholipase A(2), initiates the removal of poorly functioning mitochondria by processes involving autolysis.     

Phosphatidylinositol cleavage in lymphocytes. Requirement for calcium ions at a low concentration and effects of other cations

The soluble activity in lymphocytes which converts phosphatidylinositol into 1,2-diacylglycerol and inositol phosphates requires Ca(2+) ions. At pH7 maximum activity occurs at [Ca(2+)](free) approximately 0.7mum whereas at pH5.5 the equivalent value is approx. 50mum. At [Ca(2+)](free) approximately 1mum, a concentration similar to common intracellular values, essentially all activity is confined to the peak of activity at pH7.0. Previous reports of requirements for larger amounts of Ca(2+) may reflect the fact that the Ca(2+)-buffering capacity of phosphatidylinositol means that high substrate concentrations can effectively decrease [Ca(2+)](free). Cations which displace Ca(2+) from association with phosphatidylinositol can, at low [Ca(2+)](free), enhance enzyme activity. Phosphatidylinositol breakdown in intact cells might be controlled, at least in part, by changes in intracellular [Ca(2+)](free).     

Calcium Transport in Sealed Vesicles from Red Beet (Beta vulgaris L.) Storage Tissue : II. Characterization of Ca Uptake into Plasma Membrane Vesicles

Calcium uptake was examined in sealed plasma membrane vesicles isolated from red beet (Beta vulgaris L.) storage tissue using (45)Ca(2+). Uptake of (45)Ca(2+) by the vesicles was ATP-dependent and radiotracer accumulated by the vesicles could be released by the addition of the calcium ionophore A23187. The uptake was stimulated by gramicidin D but slightly inhibited by carbonylcyanide m-chlorophenylhydrazone. Although the latter result might suggest some degree of indirect coupling of (45)Ca(2+) uptake to ATP utilization via deltamuH(+), no evidence for a secondary H(+)/Ca(2+) antiport in this vesicle system could be found. Following the imposition of an acid-interior pH gradient, proton efflux from the vesicle was not enhanced by the addition of Ca(2+) and an imposed pH gradient could not drive (45)Ca(2+) uptake. Optimal uptake of (45)Ca(2+) occurred broadly between pH 7.0 and 7.5 and the transport was inhibited by orthovanadate, N,N'-dicyclohexylcarbodiimide, and diethylstilbestrol but insensitive to nitrate and azide. The dependence of (45)Ca(2+) uptake on both calcium and Mg:ATP concentration demonstrated saturation kinetics with K(m) values of 6 micromolar and 0.37 millimolar, respectively. While ATP was the preferred substrate for driving (45)Ca(2+) uptake, GTP could drive transport at about 50% of the level observed for ATP. The results of this study demonstrate the presence of a unique primary calcium transport system associated with the plasma membrane which could drive calcium efflux from the plant cell.     

An intracellular ATP-dependent calcium pump within the yeast Schizosaccharomyces pombe, encoded by the gene cta3.

We have permeabilized the plasma membranes of Schizosaccharomyces pombe cell with nystatin and measured ATP-dependent Ca2+ uptake in the presence of KNO3 and a protonophore in order to inhibit Ca2+ uptake into the vacuole. ATP-dependent Ca2+ accumulation into non-vacuolar Ca(2+)-storing organelles was detected. This Ca2+ uptake activity was maximal at pH 6 and inhibited by vanadate, the inhibitor of P-type ATPases. The null mutation of cta3, a putative Ca2+ gene, [Ghislain, M., Goffeau, A., Halachmi, D. and Eilam, Y. (1990) J. Biol. Chem. 265, 18400-18407] strongly reduced the level of ATP-dependent Ca2+ uptake into non-vacuolar intracellular storing organelles. This result suggests that cta3 encodes an intracellular ATP-dependent Ca2+ pump. The residual ATP-dependent Ca2+ uptake in the mutant strain indicated the presence of a second nonvacuolar, intracellular Ca(2+)-ATPase encoded by a different gene.     

Distinction between Ca(2+) pump and Ca(2+)/H(+) antiport activities in synaptic vesicles of sheep brain cortex

Synaptic vesicles, isolated from a sheep brain cortex, accumulate Ca(2+) in a manner that depends on the pH and pCa values. In the presence of 100 microM CaCl(2), most of the Ca(2+) taken up by the vesicles was vanadate-inhibited (86%) at pH 7.4, whereas at pH 8.5, part of the Ca(2+) accumulated (36%) was DeltapH-dependent (bafilomycin and CCCP inhibited) and part was insensitive to those drugs (31%). We also observed that both vanadate-sensitive and bafilomycin-sensitive Ca(2+) accumulations were completely released by the Ca(2+) ionophore, ionomycin, and that these processes work with high (K(0.5)=0.6 microM) and low (K(0.5)=217 microM) affinity for Ca(2+), respectively. The DeltapH-dependent Ca(2+) transport appears to be largely operative at Ca(2+) concentrations (>100 microM) which completely inhibited the vanadate-sensitive Ca(2+) uptake. These Ca(2+) effects on the Ca(2+) accumulation were well correlated with those observed on the vanadate-inhibited Ca(2+)-ATPase and bafilomycin-inhibited H(+)-ATPase, respectively. The Ca(2+)-ATPase activity reached a maximum at about 25 microM (pH 7.4) and sharply declined at higher Ca(2+) concentrations. In contrast, Ca(2+) had a significant stimulatory effect on the H(+)-ATPase between 250 and 500 microM Ca(2+) concentration. Furthermore, we found that DeltapH-sensitive Ca(2+) transport was associated with proton release from the vesicles. About 21% of the maximal proton gradient was dissipated by addition of 607.7 microM CaCl(2) to the reaction medium and, if CaCl(2) was present before the proton accumulation, lower pH gradients were reached. Both vanadate-inhibited and bafilomycin-inhibited systems transported Ca(2+) into the same vesicle pool of our preparation, suggesting that they belong to the same cellular compartment. These results indicate that synaptic vesicles of the sheep brain cortex contain two distinct mechanisms of Ca(2+) transport: a high Ca(2+) affinity, proton gradient-independent Ca(2+) pump that has an optimal activity at pH 7.4, and a low Ca(2+) affinity, proton gradient-dependent Ca(2+)/H(+) antiport that works maximally at pH 8.5.     

Focusing of the electrostatic potential at EF-hands of calbindin D(9k): titration of acidic residues

Biological functions for a large class of calmodulin-related proteins, such as target protein activation and Ca(2+) buffering, are based on fine-tuned binding and release of Ca(2+) ions by pairs of coupled EF-hand metal binding sites. These are abundantly filled with acidic residues of so far unknown ionization characteristics, but assumed to be essential for protein function in their ionized forms. Here we describe the measurement and modeling of pK(a) values for all aspartic and glutamic acid residues in apo calbindin D(9k), a representative of calmodulin-related proteins. We point out that while all the acidic residues are ionized predominantly at neutral pH, the onset of proton uptake by Ca(2+) ligands with high pK(a) under these conditions may have functional implications. We also show that the negative electrostatic potential is focused at the bidental Ca(2+) ligand of each site, and that the potential is significantly more negative at the N-terminal binding site.     

Studies of the Ca2+ transport mechanism of human erythrocyte inside-out plasma membrane vesicles. I. Regulation of the Ca2+ pump by calmodulin

Calcium accumulation by human erythrocyte inside-out vesicles was linear for at least 30 min in the presence of ATP. In untreated inside-out vesicles, 3.76 +/- 1.44 nmol of calcium/min/unit of acetylcholinesterase were transported, compared with 10.57 +/- 2.05 (+/- S.D.; n = 11) in those treated with calmodulin. The amount of calmodulin necessary for 50% activation of Ca2+ accumulation was 60 +/- 22 ng/ml (+/- S.D.; n = 4). The Km (Ca2+) for calmodulin-stimulated accumulation was 0.8 +/- 0.05 microM (+/- S.D.; n = 5) using Ca2+ /ethylene glycol bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA) buffers, or 25 microM with direct addition of unbuffered calcium. In the absence of calmodulin, these values were 0.4 and 60 microM, respectively, Km (ATP) values of 90 and 60 microM in the presence and absence of calmodulin, respectively, were measured at constant magnesium concentration (3 mM). In the presence of calmodulin, a broad pH profile is exhibited from pH 6.6 to 8.2. Maximal calcium accumulation occurs at pH 7.8. In the absence of calmodulin, the pH profile exhibits a linear upward increase from pH 7.0 to 8.2. The (Ca2+-Mg2+)-ATPase activity, measured under identical conditions, was 2.40 +/- 0.72 nmol of Pi/min/unit of acetylcholinesterase in the untreated vesicles and 11.29 +/- 2.87 nmol of Pi/min/unit of acetylcholinesterase (+/- S.D.; n = 4) in calmodulin-treated vesicles. A stoichiometry of 1.6 Ca2+/ATP hydrolyzed was determined in the absence of calmodulin; in the presence of calmodulin, this ratio was decreased to 0.94 Ca2+/ATP hydrolyzed.     

Dissecting the hydrolytic activities of sarcoplasmic reticulum ATPase in the presence of acetyl phosphate

Sarcoplasmic reticulum vesicles and purified Ca(2+)-ATPase hydrolyze acetyl phosphate both in the presence and absence of Ca(2+). The Ca(2+)-independent activity was fully sensitive to vanadate, insensitive to thapsigargin, and proceeded without accumulation of phosphorylated enzyme. Acetyl phosphate hydrolysis in the absence of Ca(2+) was activated by dimethyl sulfoxide. The Ca(2+)-dependent activity was partially sensitive to vanadate, fully sensitive to thapsigargin, and associated with steady phosphoenzyme accumulation. The Ca(2+)/P(i) coupling ratio at neutral pH sustained by 10 mm acetyl phosphate was 0.57. Addition of 30% dimethyl sulfoxide completely blocked Ca(2+) transport and partially inhibited the hydrolysis rate. Uncoupling induced by dimethyl sulfoxide included the accumulation of vanadate-insensitive phosphorylated enzyme. When acetyl phosphate was the substrate, the hydrolytic pathway was dependent on experimental conditions that might or might not allow net Ca(2+) transport. The interdependence of both Ca(2+)-dependent and Ca(2+)-independent hydrolytic activities was demonstrated.     

Calcium requirement for efficient phagocytosis by Dictyostelium discoideum

Extracellular EDTA suppressed in a dose-dependent manner the phagocytosis of yeast particles by Dictyostelium discoideum cells. Activity was restored fully by the addition of Ca(2+), and partially by the addition of Mn(2+)or Zn(2+), but Mg(2+)was ineffective. The pH-sensitive, Ca(2+)-specific chelator EGTA also inhibited phagocytosis at pH 7.5, but not at pH 5, and Ca(2+)restored the inhibited phagocytosis. In contrast, pinocytosis was unaffected by EDTA. Consistent with the idea that Ca(2+)was required for phagocytosis, D. discoideum growth on bacteria was inhibited by EDTA, which was then restored by the addition of Ca(2+). It is concluded that Ca(2+)was needed for efficient phagocytosis by D. discoideum amoebae. A search for Ca(2+)-dependent membrane proteins enriched in phagosomes revealed the presence of p24, a Ca(2+)-dependent cell-cell adhesion molecule-1 (DdCAD-1) that could be the target of the observed EDTA and EGTA inhibition. DdCAD-1-minus cells, however, had normal phagocytic activity. Furthermore, phagocytosis was inhibited by EDTA and rescued by Ca(2+)in the mutant just as in wild type. Thus, DdCAD-1 was not responsible for the observed Ca(2+)-dependence of phagocytosis, indicating that one or more different Ca(2+)-dependent molecule(s) was involved in the process.     

Functional Approach to the Catalytic Site of the Sarcoplasmic Reticulum Ca2+-ATPase: Binding and Hydrolysis of ATP in the Absence of Ca2+

Isolated sarcoplasmic reticulum vesicles in the presence of Mg(2+) and absence of Ca(2+) retain significant ATP hydrolytic activity that can be attributed to the Ca(2+)-ATPase protein. At neutral pH and the presence of 5 mM Mg(2+), the dependence of the hydrolysis rate on a linear ATP concentration scale can be fitted by a single hyperbolic function. MgATP hydrolysis is inhibited by either free Mg(2+) or free ATP. The rate of ATP hydrolysis is not perturbed by vanadate, whereas the rate of p-nitrophenyl phosphate hydrolysis is not altered by a nonhydrolyzable ATP analog. ATP binding affinity at neutral pH and in a Ca(2+)-free medium is increased by Mg(2+) but decreased by vanadate when Mg(2+) is present. It is suggested that MgATP hydrolysis in the absence of Ca(2+) requires some optimal adjustment of the enzyme cytoplasmic domains. The Ca(2+)-independent activity is operative at basal levels of cytoplasmic Ca(2+) or when the Ca(2+) binding transition is impeded.     

Characterization and implications of Ca2+ binding to pectate lyase C

Ca(2+) is essential for in vitro activity of Erwinia chrysanthemi pectate lyase C (PelC). Crystallographic analyses of 11 PelC-Ca(2+) complexes, formed at pH 4.5, 9.5, and 11.2 under varying Ca(2+) concentrations, have been solved and refined at a resolution of 2.2 A. The Ca(2+) site represents a new motif for Ca(2+), consisting primarily of beta-turns and beta-strands. The principal differences between PelC and the PelC-Ca(2+) structures at all pH values are the side-chain conformations of Asp-129 and Glu-166 as well as the occupancies of four water molecules. According to calculations of pK(a) values, the presence of Ca(2+) and associated structural changes lower the pK(a) of Arg-218, the amino acid responsible for proton abstraction during catalysis. The Ca(2+) affinity for PelC is weak, as the K(d) was estimated to be 0.132 (+/-0.004) mm at pH 9.5, 1.09 (+/-0.29) mm at pH 11.2, and 5.84 (+/-0.41) mm at pH 4.5 from x-ray diffraction studies and 0.133 (+/-0.045) mm at pH 9.5 from intrinsic tryptophan fluorescence measurements. Given the pH dependence of Ca(2+) affinity, PelC activity at pH 4.5 has been reexamined. At saturating Ca(2+) concentrations, PelC activity increases 10-fold at pH 4.5 but is less than 1% of maximal activity at pH 9.5. Taken together, the studies suggest that the primary Ca(2+) ion in PelC has multiple functions.