Inositol trisphosphate induces calcium release from Neurospora crassa vacuoles

Inositol 1,4,5-trisphosphate is known to release calcium ions from intracellular stores thought to be parts of endoplasmic reticulum in animal cells. In Neurospora crassa, however, inositol 1,4,5-trisphosphate acts on vacuoles stimulating a calcium efflux with a Km of 5.28 microM. The calcium release is inhibited effectively by dantrolene. These results were obtained by applying two independent methods, measuring calcium binding to fura-2 and loading vacuoles with 45Ca.     

Inositol trisphosphate stimulates calcium release from peeled skeletal muscle fibers

The effects of inositol phosphates (tris (InsP3), bis (InsP2), mono (InsP)) on rabbit adductor magnus and soleus muscles were determined using mechanically peeled fibers (sarcolemma removed). Isometric force generation of each fiber was continuously monitored and was used along with 45Ca to detect calcium release from internal fiber stores. All experiments were conducted at a physiological Mg2+ concentration (10(-3) M) of the bathing solutions. The inositol phosphates did not directly activate the contractile apparatus. At bath concentrations of 100-300 microM, only InsP3 was capable of stimulating Ca2+ release. In contrast, 1 microM InsP3 maximally and selectively stimulated Ca2+ release when microinjected into the myofilament lattice. Calcium releasing effects of InsP2 and InsP were manifested at 10 microM when they were microinjected. The end-to-end internal Ca2+ release and subsequent fiber force generation stimulated by the locally applied microinjected InsP3 suggests that the InsP3-induced Ca2+ release mechanism may involve propagation, but not via the Ca2+-induced Ca2+ release, since procaine did not inhibit this response. These findings support the possibility that InsP3 plays a role in skeletal muscle excitation-contraction coupling.     

An active proton pump of intact vacuoles isolated from Tulipa petals

Anthocyanin pigments within Tulipa petal vacuoles provide the means for real-time spectrophotometric monitoring of vacuolar sap pH and for studying ATP-dependent proton transport in isolated, intact vacuoles. Spectra of petal extracts were used to select empirically those wavelengths giving an approximately linear variation in anthocyanin absorbance with pH over a pH range of interest. A sensitive single-beam spectrophotometer with vertical optics was used to minitor absorbance changes of intact, settled vacuoles. Substrates and inhibitors of vacuolar ATPase (Lin, W., Wagner, G.J., Siegelman, H.W. and Hind, Q. (1977) Biochim. Biophys. Acta 465, 110–117) were added to probe proton transport. Acidification of the vacuole sap occurred following addition of MgATP, but not CaATP. Proton accumulation was inhibited by 10 μM Dio 9, an inhibitor of tonoplast ATPase in vitro, and the proton gradient established by addition of MgATP was dissipated after addition of 10 μM CCCP. No pumping response was observed with intact protoplasts. Potential differences across the tonoplast were directly measured by impaling vacuoles with glass microelectrodes. Potential differences of 10–20 mV (inside positive) were recorded when vacuoles were suspended in 0.7 M mannitol/10 mM Hepes buffer (adjusted to pH 8.0 with KOH), and 0.5 mM dithiothreitol. Addition of MgATP increased the potential difference by 2–5 mV.     

Transport of Ca2+ across the tonoplast of intact vacuoles from Chenopodium album L. suspension cells: ATP-dependent import and inositol-1,4,5-trisphosphate-induced release

The removal of Ca²⁺ from the medium by intact vacuoles and microsomes of Chenopodium album was investigated by measuring INDO-1 fluorescence emission at 400 and 480 nm and the response of Ca²⁺ -selective mini-electrodes. The removal of Ca²⁺ depended on the presence of MgATP, displaying an apparent K _mATP of about 50 μM, a K _mCa of 400–500 nM, and a nucleotide specificity (%) of ATP (100) > CTP (49) > GTP (28) > UTP (20) > ADP = AMP (0). In the presence of saturating MgATP, the vacuoles reduced the [Ca²⁺] of the medium below 30 nM. Part of the Ca²⁺ removed from the medium was released again after adding micromolar concentrations of inositol-1,4,5-trisphosphate. This release of Ca²⁺ was inhibited by heparin. Since digitonin caused the release of the entire amount of Ca²⁺ removed from the medium in the presence of MgATP, we argue that the Ca²⁺ is not bound to membranes or sequestered otherwise, but is transported into the vacuoles (or vesicles) and remains freely mobile there. In accordance with the current literature, we conclude that the plant vacuole is an important store for mobile Ca²⁺ to be released for purposes of signal transduction. Since changes in the trans-tonoplast ΔpH and inhibition of the H⁺-translocating pumps had no significant influence on the ATP-dependent removal of Ca²⁺ from the cytoplasmic side, we argue that in C. album ATP-driven Ca²⁺ transport is the predominant form of Ca²⁺ translocation into the vacuole. Received: 11 July 1996 / Accepted: 18 October 1996     

Phosphate uptake across the tonoplast of intact vacuoles isolated from suspension-cultured cells of Catharanthus roseus (L.) G. Don

 Transport of inorganic orthophosphate (Pi) across the tonoplast membrane was studied using intact vacuoles isolated from suspension-cultured cells of Catharanthus roseus. Orthophosphate uptake was strongly stimulated in the presence of Mg-ATP and Mg-pyrophosphate and inhibited by bafilomycin and concanamycin which are potent inhibitors of the vacuolar H⁺-ATPase. These results indicated that the build-up of an electrochemical gradient by the H⁺ pumps was essential for the uptake of Pi. Potassium thiocyanate, which dissipates the membrane potential across the tonoplast, strongly inhibited the Mg-ATP-stimulated uptake of Pi, while only a weak inhibition was observed in the presence of NH₄Cl, which dissipates the pH gradient. These results indicate that, as observed for other anions like malate or chloride, the electrical component is the driving force of Pi uptake, whereas the ΔpH plays only a minor role. Possible competitive inhibitors of Pi, MoO²⁻ ₄, VO³⁻ ₄ and CrO²⁻ ₄ were tested. Among them, CrO²⁻ ₄ strongly inhibited Pi uptake into the vacuoles. Various inhibitors of anion transport were also tested. Only 4,4-diisothiocyanostilbene-2,2′-disulfonic acid strongly inhibited Pi uptake into the vacuoles. The function of the vacuolar Pi transporters for cytoplasmic Pi homeostasis is discussed. Received: 20 September 1999 / Accepted: 28 January 2000     

Osmotic water permeability of isolated vacuoles

We measured the osmotic water permeability (P _os) of vacuoles isolated from onion (Allium cepa L.), rape (Brassica napus L.), petunia (Petunia hybrida Hook.) and red beet (Beta vulgaris L.). For all the vacuolar types investigated, P _os values were in the range 200–1000 μm s⁻¹. The change in membrane surface area induced by an osmotic gradient was smaller than 2–6%. The vacuolar P _os values for red beet and onion were reduced by 1 mM HgCl₂, to 14% and 30% of the control values, respectively, but were partially restored to 51% and 76% by 5 mM β-mercaptoethanol. These results suggest that aquaporins were present in all the vacuoles tested. In HgCl₂-treated onion vacuoles, the reduced P _os (56 μm s⁻¹) had a low activation energy (approx. 6 kJ mol⁻¹), indicating that water permeation was still occurring mainly via aquaporins, and that the water permeability of the lipid part of the vacuolar membrane is probably very low. Received: 18 February 1999 / Accepted: 21 June 1999     

Stimulation of glycogenolysis in hepatocytes by angiotensin II may involve both calcium release and calcium influx

The stimulation of [³H]glucose release (a measure of glycogenolysis) from isolated guinea pig hepatocytes by Ca-mobilizing agonists can be resolved into two phases. The initial transient phase is independent of extracellular Ca, and is probably a result of Ca released from an intracellular pool. The second phase occurs only in the presence of extracellular Ca, which suggests that Ca-influx is also involved in the mechanism of Ca-mobilization by these agents in the guinea pig hepatocyte.     

Effects of inositol 1,4,5-trisphosphate on calcium release from the endoplasmic reticulum and Golgi apparatus in mouse mammary epithelial cells: a comparison during pregnancy and lactation

It has been established that inositol 1,4,5-trisphosphate(IP3) is responsible for the mobilization of calcium(Ca2+) from intracellular locations in a wide variety of tissues, and that this response triggers the stimulation of several hormones and neurotransmitters. However, these phenomena have yet to be examined in the mammary epithelium. Ca2+ uptake from the medium into the endoplasmic reticulum(ER) and Golgi apparatus in vitro in both pregnant and lactating mouse mammary epithelial cells was studied and a strong Ca2+ release from these organelles into the medium with the use of IP3 was shown. The Ca2+ uptake and its release due to IP3 was also usually greater during pregnancy than lactation.     

Ca influx evoked by inositol-3,4,5,6-tetrakisphosphate in ras-transformed NIH/3T3 fibroblasts

Infusion of inositol-3,4,5,6-tetrakisphosphate (Ins(3,4,5,6)P₄) from the patch pipette into the cytoplasm, produced a biphasic intracellular free Ca²⁺ concentration ([Ca²⁺]_i) increase in ras-transformed NIH/3T3 (DT) cells. The Ins(3,4,5,6)P₄-induced increase in DT cells depended upon extracellular Ca²⁺ and was enhanced by membrane hyperpolarization. Identical [Ca²⁺]_i increases were observed with intracellular application of inositol-1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P₄) and inositol-1,3,4,6-tetrakisphosphate but not with inositol-1,2,4,5-tetrakisphosphate, inositol-1,4,5-trisphosphate or inositol-1,3,4,5,6-pentakisphosphate. Stimulation of DT cells with bradykinin increased the levels of Ins(3,4,5,6)P₄ and Ins(1,3,4,5)P₄. These results suggest that Ins(3,4,5,6)P₄ may serve as a second messenger for continuous Ca²⁺ influx along with other tetrakisphosphates downstream from bradykinin receptors in DT cells.     

Determination of the membrane potential of vacuoles isolated from red-beet storage tissue

The membrane potential of isolated vacuoles of red beet (Beta vulgaris L.) was estimated using several methods. The quenching of the fluorescence of the cyanine dyes 3,3-diethylthiodicarbocyanine iodide (DiS-C₂–(5)) and 3,3-dipropylthiodicarbocyanine iodide (DiS-C₃–(5)) in vacuoles indicated a transmembrane potential difference, negative inside at low external potassium concentrations. The was found to be-55 mV with two other methods, the distribution of ²⁰⁴T1⁺ in the presence of valinomycin and the distribution of the lipophilic cation triphenylmethylphosphonium. Uncouplers reduced this value to-35 mV. High external potassium concentrations, comparable to cytosolic values, abolished the membrane potential almost completely. The addition of 1 mM Tris-Mg²⁺-ATP markedly hyperpolarized the membrane to-75 mV. This effect was prevented by inhibitors of the ATPase activity located in isolated vacuole membranes.Abbreviations ANS aminonaphthalene sulfonate - DiS-C₂–(5) 3,3-diethylthiodicarbocyanine iodide - DiS-C₃–(5) 3,3-dipropylthiodicarbocyanine iodide - EDAC 1-ethyl-3-C-3dimethylaminopropylcarbodiimide - FCCP carbonylcyanide-p-trifluoromethoxyphenylhydrazone - MES morpholinoethylsulfonic acid - TPP⁺ tetraphenylphoshonium - TPMP triphenylmethylphosphonium - Tris tris(hydroxymethyl)aminomethane     

Changes in biochemical composition of vacuoles isolated from Acer pseudoplatanus L. during cell culture

Vacuoles were isolated from Acer pseudoplatanus cell suspension culture using a one-step procedure involving the lysis of the protoplast plasmalemma through a gradient of Ficoll containing DEAE-Dextran. The vacuole suspensions were slightly contaminated by other organelles (less than 5%) and the isolated vacuoles readily accumulated neutral red. Since α-mannosidase was located exclusively in the vacuoles it was used as a convenient marker. It was shown that the number of vacuoles per protoplast decreased as the cell aged. Studies on the biochemical composition of the isolated vacuoles indicated that amino acids, organic acids and protein contents varied with the cell culture cycle, emphasizing the dynamic status of the vacuolar system in cell suspension cultures of Acer pseudoplatanus.     

Hydrolysis of sucrose within isolated vacuoles from Solanum tuberosum L. tubers

The soluble acid invertase (β-D-fructofuranoside fructohydrolase, EC 3.2.1.26) from potato (Solanum tuberosum L. cv. Kennebec) tubers was located in the vacuoles. Although the functionality of this invertase in the vacuoles has been assumed, the activity of the enzyme has never been shown within isolated vacuoles. Vacuoles were prepared by gentle osmotic shock from free protoplasts obtained by enzymic digestion of tuber tissues. The mean volume of these vacuoles, (0.26 ± 0.05) × 10⁻² μl, was estimated by optical microscopy. Sucrose, glucose and fructose concentrations were calculated to be 100 mM, 20 mM and 40 mM, respectively, in the vacuoles. Sucrose hydrolysis and the increase in glucose and fructose concentrations within the vacuoles were measured during vacuolar incubations. An almost identical pattern of sucrose hydrolysis by invertase was found by an in-vitro assay reproducing the vacuolar conditions. In view of the determinations of internal vacuolar pH (5.2), the possibility of spontaneous hydrolysis of sucrose was disregarded. Vacuoles were shown to be free from proteinaceous inhibitors, confirming the extravacuolar location of these inhibitors. The vacuolar hydrolytic pattern of sucrose confirms the regulatory role of the reaction products previously proposed for in-vitro assays. Received: 21 July 1997 / Accepted: 31 August 1997     

Free calcium ion concentration in the sieve-tube sap of Ricinus communis L. seedlings

Changes in free Ca²⁺ in sieve-tube sap have been proposed to be important in the regulation of phloem transport, and Ca²⁺-activated protein kinase activity has been described in phloem exudate (S.A. Avdiushko et al. 1997 J Plant Physiol 150: 552–559). Using atomic absorption spectrometry, we have determined that the total Ca²⁺ concentration in sieve-tube sap from Ricinus seedlings containing the endosperm is about 100 μM (range 80–150 μM). We used three independent methods to determine the free calcium ion concentration in the phloem sap ([Ca²⁺]_p). The first method was to calculate [Ca²⁺]_p from the total Ca²⁺ concentration, in combination with the binding constants and concentrations of the ionic solutes in phloem sap. The resultant estimate of [Ca²⁺]_p was 63 μM. The second method used the Ca-specific fluorescent dye 2-[2-(5-carboxy)oxazole]-5-hydroxy-6-aminobenzofuran-N,N,O-triacetic-acid (FURAPTRA) on exuded sieve-tube sap. Although the sap interfered severely with the fluorescence properties of the dye, Ca²⁺ titrations enabled a value of [Ca²⁺]_p = 20 μM to be deduced. The third method used Ca²⁺-selective microelectrodes on exuded sap samples, which gave an average value for [Ca²⁺]_p = 13 μM. No significant change in this value was observed during the sap exudation period. The Ca²⁺ buffer capacity was determined and the result of about 0.6 mmol · l⁻¹ · pCa⁻¹ displayed excellent agreement with the measured values of free and total Ca²⁺ concentration in sieve-tube sap. Since the measured values for free Ca²⁺ are 20- to 100-fold higher than those usually reported for the cytosol of a range of plant cells in resting conditions, it is concluded that either regulation of [Ca²⁺]_p is of limited physiological importance, or that the Ca²⁺-dependent proteins respond only to relatively high [Ca²⁺]_p. The implications for regulation of cytosolic free Ca²⁺ in symplastically connected companion cells is discussed. Received: 15 February 1998 / Accepted: 14 March 1998     

Cell-permeant NAADP: a novel chemical tool enabling the study of Ca2+ signalling in intact cells

NAADP (nicotinic acid adenine dinucleotide phosphate) is a recently discovered second messenger, and as such, we have much yet to learn about its functions in health and disease. A bottleneck in this basic research is due to NAADP, like all second messengers, being charged to prevent it from leaking out of cells. This makes for effective biology, but imposes difficulties in experiments, as it must be injected, loaded via liposomes, or electroporated, techniques that are highly technically demanding and are possible only in certain single cell preparations. For the better understood second messenger inositol 1,4,5-trisphosphate, great success has been obtained with cell-permeant derivatives where the charged groups are masked through esterification. We now report NAADP-AM as a cell-permeant analogue of NAADP that is taken up into cells and induces NAADP-mediated Ca(2+) signalling. NAADP-AM is a powerful chemical tool that will be of enormous biological utility in a wide range of systems and will greatly facilitate research into the role of NAADP in health and disease.     

Differential coupling of alpha7 and non-alpha7 nicotinic acetylcholine receptors to calcium-induced calcium release and voltage-operated calcium channels in PC12 cells

Neuronal nicotinic acetylcholine receptors (nAChRs) are ligand-gated cation channels that can modulate various neuronal processes by altering intracellular Ca(2+) levels. Following nAChR stimulation Ca(2+) can enter cells either directly, through the intrinsic ion channel, or indirectly following voltage-operated Ca(2+) channel (VOCC) activation; Ca(2+) levels can subsequently be amplified via Ca(2+)-induced Ca(2+) release from intracellular stores. We have used subtype-selective nAChR agonists to investigate the Ca(2+) sources contributing to alpha7 and non-alpha7 nAChR-mediated increases in intracellular Ca(2+) in PC12 cells. Application of the alpha7 nAChR positive allosteric modulator PNU 120596 (10 mum), in conjunction with the alpha7 nAChR agonist, compound A [(R)-N-(1-azabicyclo[2.2.2]oct-3-yl)(5-(2-pyridyl)thiophene-2-carboxamide), 10 nm], produces a rapid increase in fluo-3 fluorescence that is prevented by the selective alpha7 nAChR antagonist alpha-bungarotoxin. The non-alpha7 nAChR agonist 5-Iodo-A-85380 produces alpha-bungarotoxin-insensitive increases in intracellular Ca(2+) (EC(50) = 11.2 mum). Using these selective agonists or KCl in conjunction with general and selective VOCC inhibitors, we demonstrate that the primary route of Ca(2+) entry following either non-alpha7 nAChR activation or KCl stimulation is via L-type VOCCs. In contrast, the alpha7 nAChR-mediated response is unaffected by VOCC blockers but is inhibited by modulators of intracellular Ca(2+) stores. These results indicate that alpha7 and non-alpha7 nAChRs are differentially coupled to Ca(2+)-induced Ca(2+) release and VOCCs, respectively.     

The vacuolar V1/V0-ATPase is involved in the release of the HOPS subunit Vps41 from vacuoles, vacuole fragmentation and fusion

At yeast vacuoles, phosphorylation of the HOPS subunit Vps41 depends on the Yck3 kinase. In a screen for mutants that mimic the yck3Delta phenotype, in which Vps41 accumulates in vacuolar dots, we observed that mutants in the V0-part of the V0/V1-ATPase, in particular in vma16Delta, also accumulate Vps41. This accumulation is not due to a phosphorylation defect, but to reduced release of Vps41 from vma16Delta vacuoles. One reason could be a connection to vacuole fission, which is blocked in V-ATPase mutants. Vacuole fusion is not impaired between vacuoles lacking the V0-subunits Vma16 or Vma6 and wild-type vacuoles, whereas fusion between mutant vacuoles is reduced. Our data suggest a connection between vacuole biogenesis and membrane fusion.     

Transport of phenylalanine into vacuoles isolated from barley mesophyll protoplasts

The energy-dependent transport of phenylalanine into isolated vacuoles of barley (Hordeum vulgare L.) mesophyll protoplasts has been studied by silicone-layer floatation filtering. The uptake of this aromatic amino acid into the vacuolar compartment is markedly increased by MgATP, showing saturation kinetics; the K _m values were 0.5 mM for MgATP and 1.2 mM for phenylalanine. V _max for phenylalanine transport was estimated to 140 nmol phenylalanine·(mg·Chl)^-1·h^-1. The transport shows a distinct pH optimum at 7.3 and is markedly inhibited by 40 mM nitrate. Azide (1 mM) and vanadate (400 M) had no or little effect on rates of transport while p-fluorophenylalanine seemed to be an effective inhibitor, indicating a possible competition at an amino-acid carrier. Ionophores such as valinomycin, nigericin or gramicidin were strong inhibitors of phenylalanine transport, indicating that this process is coupled to both the transmembrane pH gradient (pH) and the transmembrane potential ().Abbreviations and symbols BSA bovine serum albumin - Chl chlorophyll - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - pH transmembrane pH gradient - transmembrane potential     

Polypeptide pattern and enzymic character of vacuoles isolated from barley mesophyll protoplasts

Intact chloroplasts and vacuoles were isolated from mesophyll protoplasts of barley. The chloroplasts occupied about 15% of the cellular volume and contained 75% of the protein, whereas the vacuoles occupied about 80% of the volume and contained less than 4% of total cellular protein. Contamination of the vacuolar fraction by foreign protein is included in these values. Chlorophyll was absent from the vacuolar fraction, but less than 1% of several extra-vacuolar marker proteins were still present. The vacuoles contained hydrolytic enzymes. Several of them (-mannosidase, -galactosidase, N-acetylglucosaminidase) were soluble, whereas part of the activity of others semimented with the tonoplasts during centrifugation. Attached proteins could be released from the membranes during freezing in the presence of NaCl. One-dimensional gel electrophoretic separation of soluble vacuolar proteins under non-denaturing conditions yielded more than 10 protein bands. A comparative analysis was performed of thylakoids and vacuoles which were subfractionated into tonoplasts and soluble vacuolar constituents. Sodium dodecyl sulfate gel electrophoresis separated about 15 polypeptides of the soluble fraction which reacted with silver reagent. The tonoplast fraction yielded about 20 bands. A similar number of bands was observed when vacuoles incubated with the ¹⁴C-labelled SH-reagent N-ethylmaleimide were analysed for radioactive polypeptides. Silverstaining of the polypeptides and their SH-content did not correlate. Several polypeptides of the vacuolar fraction had molecular weights very similar to the molecular weights of known chloroplast proteins. However, with the exception of the two subunits of ribulose-1,5-bisphosphate carboxylase, contamination of the vacuolar fraction by chloroplast proteins could be ruled out as a possible cause of the close correspondence. The lipophilic carboxylic-group reagent N,N-dicyclohexylcarbodiimide ([¹⁴C]DCCD) reacted with several polypeptides of thylakoids and tonoplasts. However, the labelling patterns were different. The most heavily labelled polypeptide of thylakoids was the 8-kDa polypeptide of the basal part of the coupling factor CF₀. Tonoplast polypeptides heavily labelled with [¹⁴C]DCCD had molecular weights of 24, 28, and 56 kDa. The vacuolar 8-kDa polypeptide remained unlabelled.Abbreviations DCCD N,N-dicyclohexylcarbodiimide - IA iodoacetamide - NEM N-ethylmaleimide - PAGE polyacrylamide gel electrophoresis - PMSF phenylmethylsulfonylfluoride - SDS sodium dodecyl sulfate     

Requirement of an extracellular energy substrate for the guinea pig sperm acrosome reaction induced by calcium ionophore

It is well established that calcium ionophore A 23187 induces acrosome reaction (AcR) of uncapacitated spermatozoa in the presence of extracellular Ca2+ ions. In the present study, we have investigated how extracellular energy substrates (glucose, pyruvate, and lactate) affect the ionophore-induced AcR of guinea pig spermatozoa. It was found that 0.3 microM concentration of A 23187 had the maximum effect to initiate AcR of guinea pig spermatozoa. Virtually no spermatozoa underwent their AcR when incubated in substrate-free modified Tyrode's medium containing 0.3 microM A 23187 and 2 mM Ca2+. At least one exogenous substrate is essential for the ionophore-induced AcR of spermatozoa. As for efficacy of the substrates, lactate was more effective than pyruvate and glucose. However, a better result was observed when lactate was added along with pyruvate. Malonate inhibited the ionophore-induced AcR but not the hyperactivated motility of spermatozoa. The mitochondrial electron transport chain blockers rotenone, antimycin, and oligomycin failed to inhibit AcR, although in the presence of these blockers spermatozoa were unable to show hyperactivated motility. These results suggest that the mitochondrial citric acid cycle, not the electron transport chain, is probably the energy source for ionophore-induced AcR of guinea pig spermatozoa.     

Calcium influences the stability and conformation of rotavirus SAH glycoprotein VP7 expressed in Dictyostelium discoideum

We have previously reported expression of the rotavirus outer capsid glycoprotein, VP7, in the relatively new expression host, Dictyostelium discoideum. To optimise yields of recombinant VP7, we examined the role of Ca²⁺ since stability of both VP7 and mature rotavirus during a rotavirus infection are calcium-dependent. Low micromolar levels of free extracellular Ca²⁺ were required to maximise yields of VP7 in D. discoideum whilst levels of VP7 were reduced following depletion of intracellular Ca²⁺ reserves using A23187 and EGTA. Immunoblot analysis suggested that VP7 was being degraded in an intracellular compartment. Immunoprecipitation with a conformation-dependent neutralising antibody confirmed that EGTA-induced Ca²⁺ chelation alters the conformation of VP7. These results suggest that stability of VP7 is dependent on maintaining adequate levels of intracellular Ca²⁺ and that conformational changes in VP7 which occur following depletion of Ca²⁺ reserves induce rapid proteolysis of the protein. Since these results establish conditions for expressing optimal levels of VP7 in the correct conformation they have important implications for the development of a subunit vaccine based on recombinant VP7.