A comparison of calcium binding inCallinectes sapidus premolt and postmolt cuticle homogenates: Implications for regulation of biomineralization

Cuticle tissue homogenates (CTHs) fromCallinectes sapidus premolt cuticle bound approximately 367% more Ca²⁺ ions than did those from the postmolt cuticle. ThepH-stat assay which was used to comparein vitro CaCO₃ nucleation times confirmed that the premolt CTHs had greater inhibitory activity than did the postmolt CTHs. This inhibitory activity was indicated by CaCO₃ nucleation times in excess of control values. Premolt nucleation times exceeded those of postmolt samples by approximately 340%. A positive correlation was observed between Ca²⁺ binding and calcification inhibitory activity for both premolt and postmolt CTHs. Heat pretreatment of CTHs at 70°C for a 24-hr period had no significant effect on their Ca²⁺ binding. However, this heat pretreatment decreased their calcification inhibitory activity. Pretreatment of CTHs with Ca²⁺ diminished their calcification inhibitory activity. These results are consistent with a mechanism for inhibition of biocalcification by these proteins which involves their initial reversible binding to nascent calcite nuclei growth steps and kinks, rather than theirin vivo interaction with free Ca²⁺ ions in solution.     

Hemolymph ion composition and volume changes in the supralittoral isopod Ligia pallasii Brandt, during molt

We analyzed ion composition and volume of the hemolymph of Ligia pallasii in four different stages of the molt cycle using capillary electrophoresis and ³H-inulin. The main ions in the hemolymph were Na⁺, K⁺, Mg²⁺, Ca²⁺, and Cl⁻. The Ca²⁺concentration increased significantly during the molt by 47% from intermolt to intramolt and by 37% from intermolt to postmolt, probably due to resorption of Ca²⁺ from the cuticle and sternal CaCO₃ deposits. The K⁺ concentration increased significantly by 20% during molt. The hemolymph volume normalized to the dry mass of the animals decreased by 36% from intermolt to late premolt. This was due to a reduction in the hemolymph volume and to an increase in dry mass of the animals during premolt. A sudden increase in the hemolymph volume occurring between late premolt and intramolt served to expand the cuticle. Since the Na⁺, K⁺, Mg²⁺, and Cl⁻ concentrations did not change significantly from late premolt to intramolt, the increase in hemolymph volume suggests an uptake of seawater rather than freshwater. Accepted: 7 March 2000     

Novel protein inhibits in vitro precipitation of calcium carbonate.

Organic molecules both coexist and interact with inorganic crystal lattices in biomineralizing tissues. Mineral precipitation and crystal morphology are tightly regulated by the actions of these molecules. Polyacrylamide gel electrophoresis studies on water soluble extracts from the cuticle of Callinectes sapidus (Atlantic blue crab) reveal the presence, in unmineralized nascent premolt cuticle, of proteins which are absent in the mineralized postmolt cuticle. In the present studies, homogenates from both premolt and postmolt C. sapidus cuticles have been tested for their effect on the in vitro precipitation of calcium carbonate. The role of protein in this process was determined by heat pretreatment and trypsin pretreatment of the cuticle homogenates prior to the precipitation assay. The results from these experiments indicate that proteins, with molecular weights of approximately 75,000 and between 10,000 and 20,000, concentrated in the C. sapidus premolt cuticle, inhibit calcium carbonate precipitation in vitro. The inhibitory activity of these proteins appears to be a result of specific interactions since trypsin, myoglobin, and ovalbumin are not inhibitory. The presence of lower amounts of these inhibitory proteins in C. sapidus postmolt cuticle may be responsible for the subsequent mineralization of this tissue.     

Bacterial Calcium Carbonate Precipitation in Cave Environments: A Function of Calcium Homeostasis

To determine if microbial species play an active role in the development of calcium carbonate (CaCO ₃ ) deposits (speleothems) in cave environments, we isolated 51 culturable bacteria from a coralloid speleothem and tested their ability to dissolve and precipitate CaCO ₃ . The majority of these isolates could precipitate CaCO ₃ minerals; scanning electron microscopy and X-ray diffractrometry demonstrated that aragonite, calcite and vaterite were produced in this process. Due to the inability of dead cells to precipitate these minerals, this suggested that calcification requires metabolic activity. Given growth of these species on calcium acetate, but the toxicity of Ca ²⁺ ions to bacteria, we created a loss-of-function gene knock-out in the Ca ²⁺ ion efflux protein ChaA. The loss of this protein inhibited growth on media containing calcium, suggesting that the need to remove Ca ²⁺ ions from the cell may drive calcification. With no carbonate in the media used in the calcification studies, we used stable isotope probing with C ¹³ O ₂ to determine whether atmospheric CO ₂ could be the source of these ions. The resultant crystals were significantly enriched in this heavy isotope, suggesting that extracellular CO ₂ does indeed contribute to the mineral structure. The physiological adaptation of removing toxic Ca ²⁺ ions by calcification, while useful in numerous environments, would be particularly beneficial to bacteria in Ca ²⁺ -rich cave environments. Such activity may also create the initial crystal nucleation sites that contribute to the formation of secondary CaCO ₃ deposits within caves.     

Influencing factors and formation mechanism of CaCO3 precipitation induced by microbial carbonic anhydrase

Microbial carbonic anhydrase promotes carbonate deposition, which is important in the formation and evolution of global carbon cycle and geological processes. A kind of bacteria producing extracellular carbonic anhydrase was selected to study the effects of temperature, pH value and Ca²⁺ concentration on bacterial growth, carbonic anhydrase activity and calcification rate in this paper. The results showed that the activity of carbonic anhydrase at 30 °C was the highest, which was beneficial to the calcification reaction, calcification rate of CaCO₃ was the fastest in alkaline environment with the initial pH value of 9.0. When the Ca2+ concentration was 60 mM, compared with other Ca²⁺ concentration, CA bacteria could grow and reproduce best, and the activity of bacteria was the highest, too low Ca²⁺ concentration would affect the generation of CaCO₃, while too high Ca²⁺ concentration would seriously affect the growth of bacteria and reduce the calcification rate. Finally, the mechanism of CaCO₃ precipitation induced by microbial carbonic anhydrase was studied. Carbonic anhydrase can accelerate the hydration of CO₂ into HCO₃⁻, and react with OH⁻ and Ca²⁺ to form CaCO₃ precipitation in alkaline environment and in the presence of calcium source.     

Photosynthesis,Respiration and Exopolymer Calcium-Binding in Biofilm Calcification (Westerhöfer and Deinschwanger Creek,Germany)

The impact of microbial activity on biofilm calcification in aquatic environments is still a matter of debate, especially in settings where ambient water has high CaCO₃ mineral supersaturation. In this study, biofilms of two CO₂-degassing karst-water creeks in Germany, which attain high calcite supersaturation during their course downstream, were investigated with regard to water chemistry of the biofilm microenvironment. The biofilms mainly consisted of filamentous cyanobacteria (Phormidium morphotype) and heterotrophic bacteria (including sulfate-reducing bacteria), which affect the microenvironment and produce acidic exopolymers. In situ and ex situ microelectrode measurements showed that a strong pH increase, coupled with Ca^{2 +} consumption, occurred in light conditions at the biofilm surface, while the opposite occurred in the dark. Calcite supersaturation at the biofilm surface, calculated from ex situ Ca^{2 +} and CO₃ ^2? microelectrode measurements, showed that photosynthesis resulted in high omega values during illumination, while respiration slightly lowered supersaturation values in the dark, compared to values in the water column. Dissociation calculation demonstrated that the potential amount of Ca^{2 +} binding by exopolymers would be insufficient to explain the Ca^{2 +} loss observed, although Ca^{2 +} complexation to exopolymers might be crucial for calcite nucleation. No spontaneous precipitation occurred on biofilm-free limestone substrates under the same condition, regardless of high supersaturation. These facts indicate that photosynthesis is a crucial mechanism to overcome the kinetic barrier for CaCO₃ precipitation, even in highly supersaturated settings.     

The ionic hemolymph composition of the terrestrial isopod Porcellio scaber Latr. during molt

We analyzed the ionic composition of the hemolymph of Porcellio scaber in four different stages of the molt cycle using capillary electrophoresis and calcium selective mini- and microelectrodes. The main ions in the hemolymph were K⁺, Ca²⁺, Na⁺, Mg⁺, and Cl⁻. The values for total calcium obtained by means of capillary electrophoresis and calcium selective minielectrodes did not differ significantly from each other. In situ measurements of the free Ca²⁺ concentration ([Ca²⁺]) by means of calcium-selective microelectrodes indicated that Ca²⁺ is not bound in the hemolymph. During molt the [Ca²⁺] is significantly larger than during intermolt. The [Ca²⁺] increased by 13%, 19% and 18% during premolt, intramolt, and postmolt, respectively. The concentration of the other cations and of Cl⁻ decreased significantly between premolt and intramolt. Thus, the rise of the [Ca²⁺] in the hemolymph is not due to a general increase in all ions, but rather to the resorption of cuticular calcium. Furthermore, the results suggest that K⁺, Na⁺, Mg⁺, and Cl⁻are extruded from the hemolymph during and/or after posterior ecdysis. Accepted: 5 August 1997     

Cloning and characterization of a calmodulin gene (CaM) in crayfish Procambarus clarkii and expression during molting

Calmodulin (CaM) is a highly conserved calcium (Ca²⁺) binding protein that transduces Ca²⁺ signals into downstream effects influencing a range of cellular processes, including Ca²⁺ homeostasis. The present study explores CaM expression when Ca²⁺ homeostasis is challenged during the mineralization cycle of the freshwater crayfish (Procambarus clarkii). In this paper we report the cloning of a CaM gene from axial abdominal crayfish muscle (referred to as pcCaM). The pcCaM mRNA is ubiquitously expressed but is far more abundant in excitable tissue (muscle, nerve) than in any epithelia (gill, antennal gland, digestive) suggesting that it plays a greater role in the biology of excitation than in epithelial ion transport. In muscle cells the pcCaM was colocalized on the plasma membrane with the Ca²⁺ ATPase (PMCA) known to regulate intracellular Ca²⁺ through basolateral efflux. While PMCA exhibits a greater upregulation in epithelia (than in non-epithelial tissues) during molting stages requiring transcellular Ca²⁺ flux (pre- and postmolt compared with intermolt), expression of pcCaM exhibited a uniform increase in epithelial and non-epithelial tissues alike. The common increase in expression of CaM in all tissues during pre- and postmolt stages (compared with intermolt) suggests that the upregulation is systemically (hormonally) mediated. Colocalization of CaM with PMCA confirms physiological findings that their regulation is linked.     

Calcium-dependent cyclic nucleotide phosphodiesterase inhibition of basal activity by heat-stable factors from rat cerebrum

The boiled supernatant fraction from rat cerebrum contained factors which inhibited the basal activity of a Ca²⁺-dependent phosphodiesterase from rat cerebrum. Two inhibitory fractions were isolated by DEAE-cellulose or Sephadex chromatography and were deemed proteins, based on their sensitivity to trypsin digestion. The inhibitory fractions eluted from DEAE-cellulose columns prior to the Ca²⁺-dependent activator protein. The inhibitory factors, unlike the activator protein, were stable to heat treatment under alkaline conditions. The inhibitory factors caused both an increase in K_m for cyclic GMP and a decrease in $\text{V}$. In the presence of calcium ions and purified activator protein, the Ca²⁺-dependent phosphodiesterase was not inhibited by the factors, but instead was slightly stimulated. The inhibitory factors caused a slight apparent stimulation of a Ca²⁺-independent phosphodiesterase from rat cerebrum but this proved instead to be a nonspecific stabilizing effect which was mimicked by bovine serum albumin. After prolonged alkaline treatment, the purified activator protein caused a modest Ca²⁺-independent activation of Ca²⁺-dependent phosphodiesterase. The inhibitory factors antagonized the activation of Ca²⁺-dependent phosphodiesterase by alkaline treated activator protein or by lysophosphatidylcholine. The inhibitory factors had no effect on activity of trypsinized Ca²⁺-dependent phosphodiesterase. Of various other proteins, only casein mimicked the effects of the inhibitory factors on phosphodiesterase activity.     

The cationic composition and pH in the moulting fluid of Porcellio scaber (Crustacea, Isopoda) during calcium carbonate deposit formation and resorption

Before moulting, terrestrial isopods resorb calcium carbonate (CaCO₃) from the posterior cuticle and store it in sternal deposits. These consist mainly of amorphous calcium carbonate (ACC) spherules that develop within the ecdysial space between the anterior sternal epithelium and the old cuticle. Ions that occur in the moulting fluid, including those required for mineral deposition, are transported from the hemolymph into the ecdysial space by the anterior sternal epithelial cells. The cationic composition of the moulting fluid probably affects mineral deposition and may provide information on the ion-transport activity of the sternal epithelial cells. This study presents the concentrations of inorganic cations within the moulting fluid of the anterior sternites during the late premoult and intramoult stages. The most abundant cation is Na⁺ followed by Mg²⁺, Ca²⁺ and K⁺. The concentrations of these ions do not change significantly between the stages whereas the mean pH changed from 8.2 to 6.9 units between mineral deposition in late premoult, and resorption in intramoult, respectively. Measurements of the transepithelial potential show that there is little driving force for passive movements of calcium across the anterior sternal epithelium. The results suggest a possible role of magnesium ions in ACC formation, and a contribution of pH changes to CaCO₃ precipitation and dissolution.     

Exact Stochastic Simulation of a Calcium Microdomain Reveals the Impact of Ca2+ Fluctuations on IP3R Gating

In this study, we numerically analyzed the nonlinear Ca²⁺-dependent gating dynamics of a single, nonconducting inositol 1,4,5-trisphosphate receptor (IP₃R) channel, using an exact and fully stochastic simulation algorithm that includes channel gating, Ca²⁺ buffering, and Ca²⁺ diffusion. The IP₃R is a ubiquitous intracellular Ca²⁺ release channel that plays an important role in the formation of complex spatiotemporal Ca²⁺ signals such as waves and oscillations. Dynamic subfemtoliter Ca²⁺ microdomains reveal low copy numbers of Ca²⁺ ions, buffer molecules, and IP₃Rs, and stochastic fluctuations arising from molecular interactions and diffusion do not average out. In contrast to models treating calcium dynamics deterministically, the stochastic approach accounts for this molecular noise. We varied Ca²⁺ diffusion coefficients and buffer reaction rates to tune the autocorrelation properties of Ca²⁺ noise and found a distinct relation between the autocorrelation time τ_ac, the mean channel open and close times, and the resulting IP₃R open probability P_O. We observed an increased P_O for shorter noise autocorrelation times, caused by increasing channel open times and decreasing close times. In a pure diffusion model the effects become apparent at elevated calcium concentrations, e.g., at [Ca²⁺] = 25 μM, τ_ac = 0.082 ms, the IP₃R open probability increased by ≈20% and mean open times increased by ≈4 ms, compared to a zero noise model. We identified the inactivating Ca²⁺ binding site of IP₃R subunits as the primarily noise-susceptible element of the De Young and Keizer model. Short Ca²⁺ noise autocorrelation times decrease the probability of Ca²⁺ association and consequently increase IP₃R activity. These results suggest a functional role of local calcium noise properties on calcium-regulated target molecules such as the ubiquitous IP₃R. This finding may stimulate novel experimental approaches analyzing the role of calcium noise properties on microdomain behavior.     

The effect of metal ions on the amidolytic activity of human factor Xa (Activated Stuart-Prower Factor)

The effect of metal ions on human activated Factor X (Factor X_a) hydrolysis of the chromogenic substrate benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide (S2222) was studied utilizing initial rate enzyme kinetics. The divalent metal ions Ca²⁺, Mn²⁺, and Mg²⁺ enhanced Factor X_a amidolytic activity with K_m values of 30 μm, 20 μm, and 1.4 mm, respectively. Na⁺ activation of Factor X_a amidolytic activity was also found. The K_m for Na⁺ activation was 0.31 m. Both the divalent metal ions and Na⁺ increased the affinity of Factor X_a for S2222 and had no effect on the maximal velocity of the reaction. Other monovalent cations were unable to activate Factor X_a. However, K⁺ was a competitive inhibitor of the Na⁺ activation (K_i = 0.14 m). Lanthanide ions inhibited Factor X_a amidolytic activity. Gd³⁺ inhibition of Factor X_a hydrolysis of S2222 was noncompetitive and had a K_i of 3 μm. The lanthanide ion inhibition could not be reversed by Ca²⁺ even when Ca²⁺ was present in a 1000-fold excess over its K_m indicating nonidentity of the Factor X_a lanthanide and Ca²⁺ binding sites. It is concluded that the Factor X_a Ca²⁺ binding sites have characteristics different from those previously described for the Factor X molecule and that Mg²⁺, Na⁺, and K⁺ may be physiological regulators of Factor X_a activity.     

Modeling Ca2+ feedback on a single inositol 1,4,5-trisphosphate receptor and its modulation by Ca2+ buffers

The inositol 1,4,5-trisphosphate receptor/channel (IP₃R) is a major regulator of intracellular Ca²⁺ signaling, and liberates Ca²⁺ ions from the endoplasmic reticulum in response to binding at cytosolic sites for both IP₃ and Ca²⁺. Although the steady-state gating properties of the IP₃R have been extensively studied and modeled under conditions of fixed [IP₃] and [Ca²⁺], little is known about how Ca²⁺ flux through a channel may modulate the gating of that same channel by feedback onto activating and inhibitory Ca²⁺ binding sites. We thus simulated the dynamics of Ca²⁺ self-feedback on monomeric and tetrameric IP₃R models. A major conclusion is that self-activation depends crucially on stationary cytosolic Ca²⁺ buffers that slow the collapse of the local [Ca²⁺] microdomain after closure. This promotes burst-like reopenings by the rebinding of Ca²⁺ to the activating site; whereas inhibitory actions are substantially independent of stationary buffers but are strongly dependent on the location of the inhibitory Ca²⁺ binding site on the IP₃R in relation to the channel pore.     

The effect of calcium (II) on the binding of anticoagulation factor I with activated coagulation factor X

Anticoagulation factor I (ACF I) from the venom of Agkistrodon acutus forms a 1:1 complex with activated coagulation factor X (FXa) in a Ca²⁺-dependent fashion and thereby prolongs the clotting time. In the present study, the dependence of the binding of ACF I with FXa on the concentration of Ca²⁺ ions was quantitatively analyzed by HPLC, and the result showed that the maximal binding of ACF I to FXa occurred at concentration of Ca²⁺ ions of about 1 mM. The binding of Ca²⁺ ions to ACF I was investigated by equilibrium dialysis and two Ca²⁺-binding sites with different affinities were identified. At pH 7.6, the apparent association constants K₁ and K₂ for these two sites were (1.8 ± 0.5) × 10⁵ and (2.7 ± 0.6) × 10⁴ M^–1 (mean ± SE, n = 4), respectively. It was evident from the observation of Ca²⁺-induced changes in the intrinsic fluorescence of ACF I that ACF I underwent a conformational change upon binding of Ca²⁺ ions. The occupation of both Ca²⁺-binding sites in ACF I required a concentration of Ca²⁺ ions of about 1 mM, which is equal to the effective concentration of Ca²⁺ ions required both for maximal binding of ACF I to FXa and for the maximal enhancement of emission fluorescence of ACF I. It could be deduced from these results that the occupation of both Ca²⁺-binding sites in ACF I with Ca²⁺ ions and subsequent conformational rearrangement might be essential for the binding of ACF I to FXa.     

Synaptic plasticity and humoral modulation of neuromuscular transmission in the lobster claw opener during the molt cycle

1. Neuromuscular properties of the lobster dactyl opener were studied at different stages of the molt cycle.2. Excitatory junctional potentials (EJPs) were found to be significantly larger in hard-shelled premolt lobsters than in soft-shelled postmolt animals.3. Inhibitory junctional potentials (IJPs) were larger in postmolt claw preparations than in premolt ones.4. Excitatory transmission was also monitored during superfusion with plasma samples obtained from lobsters in vartious stages of the molt cycle (postmolt = AB; intennolt = C; premolt = D₂).5. D₂ plasma increased EJP amplitude by an average of 28% relative to baseline levels (in saline), while AB plasma reduced EJP size by an average of 6%.6. Premolt and postmolt plasma produced opposite effects on quantal content (m); D₂; plasma increased m by a mean of 39%, whereas AB plasma caused a mean reduction by 10%, which suggests that humoral factors may act presynaptically to alter transmitter release.7. These results provide evidence of neuromuscular plasticity in the opener muscle during the molt cycle, and are consistent with changes in claw-opening behavior seen during this cycle.     

Identification of glycogen as the major xylose acceptor in polysomal preparations from chick embryo cartilage cultures

The capacity of various metal ions to support activation of bovine factor IX, by the coagulant protein of Russell's Viper venom, has been examined. The following metal ions, at concentrations which saturate their effect, promoted activation of factor IX, at approximately equal efficiency: Ca²⁺, Mn²⁺, Sr²⁺, and Co²⁺, Other metal ions, i.e., Ba²⁺, and Mg²⁺, at saturating concentrations, led to a maximum rate of activation of factor IX of 25%, compared to Ca²⁺, The lanthanides, Gd²⁺, and Tb³⁺, also promoted activation in this system, at maximal rates of approximately 15%, compared to Ca²⁺, In this study, it was also discovered that the esterase activity of bovine factor IXa was dependent upon the presence of metal ions. Utilizing α-N-benzoyl-l-arginine ethyl ester as the substrate, steady state kinetic analysis in the absence of metal ion indicated that the K_m and V_max for this substrate was 20 mm and 2.9 μmol substrate cleaved min^?1 mg^?1 of factor IXa, respectively, at pH 8.0 and 30 °C. In the presence of optimal concentrations of Ca²⁺, Mn²⁺, Mg²⁺, Sr²⁺, and Ba²⁺, the V_max values for this same substrate increased to 6.7, 5.9, 5.0, 5.0, and 3.7 μmol cleaved min^?1 mg^?1 of factor IXa, respectively. None of these metal ions had an affect on the K_m value of this substrate.     

Effects of CuCl2 on the hydrolysis of cyclic GMP and cyclic AMP by the activator-dependent cyclic nucleotide phosphodiesterase from bovine heart

CuCl₂ non-comepetitively inhibited the hydrolysis of cyclic GMP and cyclic AMP by the activator-dependent phosphodiesterase from bovine heart in the presence of 5 mM Mg²⁺, 10 μM Ca²⁺ and phosphodiesterase activator with K_i values of approximately 2 μM for both substrates. CuCl₂ inhibition was also non-competitive with Mg²⁺, Ca²⁺ and phosphodiesterase activator. Dialysis demonstrated that CuCl₂ inhibition in reversible. Treatment of the enzyme with p-hydroxymercuribenzoate resulted in the loss of enzyme activity, suggesting the presence of sulfhydryl groups essential for enzyme activity. The inhibitory activity of CuCl₂ was not additive with that p-hydroxymercuribenzoate, therefore CuCl₂ may inhibit enzyme activity by binding to one or more essential sulfhydryl groups. CuCl₂ also inhibited the hydrolysis of cyclic AMP by the cyclic AMP-specific phosphodiesterase from bovine heart with an I₅₀ value of 18 μM. Several effects of Cu²⁺ are discussed which have been noted in other studies and might be due, in part, to changes in cyclic nucleotide levels following alterations in phosphodiesterase activity.     

Regulation of vacuolar h-pyrophosphatase by free calcium : a reaction kinetic analysis

The H⁺-translocating inorganic pyrophosphatase (H⁺-PPase) associated with vesicles of the vacuolar membrane (tonoplast) isolated from beet (Beta vulgaris L.) is subject to direct inhibition by Ca²⁺ and a number of other divalent cations (Co²⁺, Mn²⁺, Zn²⁺). By contrast, the H⁺-translocating ATPase (H⁺-ATPase) located on the same membrane is insensitive to Ca²⁺. Here we examine the mechanism and feasibility of regulation of the vacuolar H⁺-PPase by cytosolic free Ca²⁺ under the conditions thought to prevail in vivo with respect to Mg²⁺, inorganic pyrophosphate (PPi), and pH. The minimal reaction scheme that satisfactorily describes the effects of elevated Ca²⁺ or CaPPi on the enzyme is one that invokes equilibrium binding of substrate (Mg₂PPi) at one site, inhibitory binding of Mg₂PPi to a lower-affinity second site, binding of activator (Mg²⁺) at a third site, and direct binding of Ca²⁺ or CaPPi to a fourth site. Changes in enzyme activity in response to selective manipulation of either Ca²⁺ or CaPPi are explicable only if Ca²⁺, rather than CaPPi, is the inhibitory ligand. This conclusion is supported by the finding that CaPPi fails to mimic substrate in protection of the enzyme from inhibition by N-ethylmaleimide. Furthermore, the reaction scheme quantitatively and independently predicts the observed noncompetitive effects of free Ca²⁺ on the substrate concentration dependence of H⁺-PPase activity. The results are discussed in relation to the previous proposal that CaPPi is the principal inhibitory ligand of the vacuolar H⁺-PPase (M. Maeshima [1991] Eur J Biochem 196: 11-17) and the possibility that in vivo modulation of cytosolic free Ca²⁺ might constitute a specific mechanism for selective regulation of this enzyme, and consequently for stabilization of PPi levels in the cytoplasm of plant cells.     

Functional Roles of the Non-Catalytic Calcium-Binding Sites in the N-Terminal Domain of Human Peptidylarginine Deiminase 4

This study investigated the functional roles of the N-terminal Ca²⁺ ion-binding sites, in terms of enzyme catalysis and stability, of peptidylarginine deiminase 4 (PAD4). Amino acid residues located in the N-terminal Ca²⁺-binding site of PAD4 were mutated to disrupt the binding of Ca²⁺ ions. Kinetic data suggest that Asp155, Asp157 and Asp179, which directly coordinate Ca3 and Ca4, are essential for catalysis in PAD4. For D155A, D157A and D179A, the k _cat/K _m,BAEE values were 0.02, 0.63 and 0.01 s⁻¹mM⁻¹ (20.8 s⁻¹mM⁻¹ for WT), respectively. Asn153 and Asp176 are directly coordinated with Ca3 and indirectly coordinated with Ca5 via a water molecule. However, N153A displayed low enzymatic activity with a k _cat value of 0.3 s⁻¹ (13.3 s⁻¹ for wild-type), whereas D176A retained some catalytic power with a k _cat of 9.7 s⁻¹. Asp168 is the direct ligand for Ca5, and Ca5 coordination by Glu252 is mediated by two water molecules. However, mutation of these two residues to Ala did not cause a reduction in the k _cat/K _m,BAEE values, which indicates that the binding of Ca5 may not be required for PAD4 enzymatic activity. The possible conformational changes of these PAD4 mutants were examined. Thermal stability analysis of the PAD4 mutants in the absence or presence of Ca²⁺ indicated that the conformational stability of the enzyme is highly dependent on Ca²⁺ ions. In addition, the results of urea-induced denaturation for the N153, D155, D157 and D179 series mutants further suggest that the binding of Ca²⁺ ions in the N-terminal Ca²⁺-binding site stabilizes the overall conformational stability of PAD4. Therefore, our data strongly suggest that the N-terminal Ca²⁺ ions play critical roles in the full activation of the PAD4 enzyme.