Development of Sea Urchin Embryos in Artificial Sea Water Containing Br−in Place of Cl−

In artificial sea water in which the Cl⁻concentration was reduced to less than 10% of that in normal sea water by its replacement with Br⁻, sea urchin eggs were fertilized and developed into abnormal plutei following almost the same time schedule as in natural sea water. These embryos had poorly developed spicules, short pluteus arms, somewhat jagged embryo-walls and quasi-normal archenterons. Similar embryos were obtained in another artificial sea water in which 90% of the Cl⁻concentration in normal sea water was reduced by Br⁻and 10% by acetate. In artificial sea water, in which either 90% of the Cl⁻was replaced by Br⁻or 10% was replaced by acetate, embryos developed into plutei with quasi-normal spicules, pluteus arms and archenterons. These findings indicate that deficiency of Cl⁻results in somewhat abnormal sea urchin embryos. When cells derived from isolated micromeres, were cultured in these Cl⁻-deficient artificial sea waters, containing Br⁻in place of more than 70% of the normal Cl⁻concentration in sea water, spicule formation was strongly inhibited, but pseudopodial cables were well developed. Thus, external Cl⁻seems to be necessary for at least normal formation of spicule rods.     

Spicule Formation in the Invertebrates with Special Reference to the Gorgonian Leptogorgia virgulata

Many of the invertebrates possess calcium carbonate spicules.This paper is a review of the formation of these structuresin the Porifera, Coelenterata, Platyhelminthes, Mollusca, Echinodermataand Ascidiacea. Mature spicules appear to be extracellular structures.Sponge spicules initiate intercellularly then become extracellular.Alcyonarian, turbellarian, echinoid and ascidian spicule depositionbegins intracellularly and then becomes extracellular. The continuationof growth in the extracellular environment has not been documentedexcept for the echinoids. Placophoran spicules initiate andremain as extracellular structures. Early spicule growth seemsto occur from or within a single cell. However, cell aggregationand/or neighboring cells appear to be important to the processof spicule formation. The spicule forming cells, in general,are found in a collagenous medium which may be associated withspicule growth. The organic matrix from the spicules of the gorgonian Leptogorgiavirgulata is a glycoprotein. Autoradiography reveals that thismatrix is apparently synthesized in the rough endoplasmic reticulumand Golgi complexes and then transported to the spicule formingvacuole via Golgi vesicles. To gain information about the entryand transport of calcium ions, the effects of ouabain and vanadateon calcium uptake were examined. Ouabain had no effect on calciumuptake. Vanadate treatment increased the uptake of calcium inscleroblasts and epithelial tissue and decreased its uptakein spicules. This may suggest that vanadate sensitive ATPasesare involved in the pumping of calcium out of scleroblasts,out of epithelial cells into the mesoglea, and into scleroblastorganelles. Autoradiography using ⁴⁵Ca indicates that the majorityof these ions initially accumulate in the branch axis. The labelmoves through the axial epithelium to the mesoglea and reachesthe spiculeforming vacuoles in the scleroblasts via dense bodies     

Role of ABH blood group antigens in the stimulation of a DIDS-sensitive Ca2+ influx pathway in human erythrocytes by Ulex europaeus agglutinin I and a monoclonal anti A1 antibody

Of eleven agglutinating lectins tested, only one, Ulex europaeus agglutinin I (UEA1), stimulated Ca2+ uptake in quin2-loaded erythrocytes by about 2-fold. UEA1 is known to be an alpha-L-fucose and ABH blood group specific lectin. The 45Ca2+ influx induced by UEA1 was absent in the presence of extracellular fucose (5 and 15 mM) and depended on the ABH blood group of the donor, the stimulatory potency of the lectin decreasing in the order H greater than A2 greater than A1. Ca2+ entry blockers, such as cobalt and verapamil, did not affect the 45Ca2+ influx induced by UEA1. 4,4'-Diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) inhibited dose-dependently with a Ki of 1-2 microM. 10 microM DIDS, 10 microM 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS) and 20 microM dipyridamole fully blocked the 45Ca2+ influx induced by UEA1. The effect of UEA1 on 45Ca2+ influx was absent in K+ and Mg2+ media and was less pronounced in choline than in Na+ media. The 45Ca2+ influx induced by the lectin was abolished by preincubation with 12-O-tetradecanoylphorbol 13-acetate (TPA, 60 ng/ml). A monoclonal antibody raised against A1 erythrocytes (Bric 54) accelerated 45Ca2+ influx in quin2 loaded A1 erythrocytes by about 2-fold. No effect was seen in A2 and H erythrocytes. The 45Ca2+ influx elicited by Bric 54 exhibited a sensitivity towards inhibition by DIDS and TPA, as well as a dependence on the cation composition of the incubation medium similar to that observed with UEA1. The effects of UEA1 and Bric 54 were not additive. These observations suggest that the Ca2+ influx induced by UEA1 and Bric 54 is mediated by the same transport pathway. Since both the lectin and the antibody exhibit ABH blood group specificity, it appears reasonable to conclude that ABH antigens can serve as recognition sites for activation of a Ca2+ influx pathway in human erythrocytes, which is sensitive to inhibitors of the band 3 anion-exchanger.     

Extracellular ATP induces ion fluxes and inhibits growth of Friend erythroleukemia cells

Extracellular ATP (1 mM) inhibited the growth of Friend virus-infected murine erythroleukemia cells (MEL cells) but had no effect on dimethyl sulfoxide-induced differentiation. ATP (1 mM) also caused changes in the permeability of MEL cells to ions. There was an increased influx of 45Ca2+ from a basal level of 5 pmol/min to 18 pmol/min/10(6) cells to achieve a 2-fold increase in steady-state Ca2+ as measured at isotopic equilibration. Ca2+ influx was blocked by diisothiocyanostilbene disulfonate (DIDS), an inhibitor of anion transport. ATP also stimulated Cl- uptake, and this flux was inhibited by DIDS. The ratio of ATP stimulated Cl- to Ca2+ uptake was 1.6:1. K+ and Na+ influx were also stimulated by ATP, but phosphate uptake was inhibited; the Na+ influx dissipated the Na+ gradient and thus inhibited nutrient uptake. ATP-stimulated K+ influx was ouabain inhibitable; however, the total cellular K+ decreased due to an ATP-stimulated ouabain-resistant K+ efflux. Na+ influx and Ca2+ influx occurred by separate independent routes, since Na+ influx was not inhibited by DIDS. The effects observed were specific for ATP *K1/2 MgATP = 0.7 mM) since AMP, GTP, adenosine, and the slowly hydrolyzable ATP analogue adenyl-5'-yl imidodiphosphate were without effect. The major ionic changes in the cell were a decrease in K+ and increase in Na+; cytoplasmic pH and free Ca2+ did not change appreciably. These ATP-induced changes in ion flux are considered to be responsible for growth inhibition.     

The effects of several ion channel blockers and calmodulin antagonists on fertilization-induced acid release and 45Ca2+ uptake in sea urchin eggs

The effects of calcium antagonists, diltiazem and verapamil, and calmodulin antagonists, chlorpromazine, N-(6-aminohexyl)-1-naphthalenesulfonamide hydrochloride (W-5) and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7), were tested on two responses of the sea urchin egg to insemination: (1) H+ release; (2) Ca2+ uptake. It was found that calcium antagonists inhibited both processes, while calmodulin antagonists only inhibited H+ release but not Ca2+ uptake. Verapamil and diltiazem were effective to inhibit H+ release when added to the egg suspension up to 120 sec and W-7 was effective around 150 sec after insemination. Calcium antagonists became ineffective earlier than W-7 in inhibiting H+ release. A calmodulin-dependent step may thus occur linking the Ca2+ uptake and H+ release. 4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), an anion channel blocker, also inhibited both Ca2+ uptake and H+ release. This result suggests that an uptake of anion(s) occurs along with Ca2+ uptake.     

Inhibitory Effect of Omeprazole, a Specific Inhibitor of H+, K+-ATPase, on Spicule Formation in Sea Urchin Embryos and in Cultured Micromere-Derived Cells.

Embryos kept with omeprazole, a specific H⁺, K⁺-ATPase inhibitor, in a period of development between the mesenchyme blastula and the pluteus corresponding stage became abnormal plutei having quite small spicules, somewhat poor pluteus arms and apparently normal archenterons. In micro-mere-derived cells, kept with omeprazole at pH 8.2 in a period between 15 and 40 hr of culture at 20°C, omeprazole strongly inhibited spicule formation but did not block the outgrowth of pseudopodial cables, in which spicule rods were to be formed. These indicate that omeprazole probably exerts no obvious inhibitory effects other than spicule rods formation. Omeprazole-sensitive H⁺, K⁺-ATPase, an H⁺pump, seems to be indispensable for CaCO₃ deposition (formation of spicule rod) in these spicule forming cells. H⁺, produced in overall reaction for CaCO₃ formation: Ca²⁺+ CO₂+H₂O°CaCO₃+2H⁺, is probably released from the cells by this H⁺pump and hence, this reaction tends to go to CaCO₃ production to form spicule rods. Omeprazole, known to become effective following its conversion to a specific inhibitor of H⁺, K⁺-ATPase at acidic pH, is able to inhibit formation of spicule rod at alkaline pH in sea water. This is probably due to an acidification of sea water near the cell surface by H⁺ejection in H⁺, K⁺-ATPase reaction.     

Evidence that the erythrocyte calcium pump catalyzes a Ca2+:nH+ exchange

Treatment of whole erythrocytes with 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS) results in inhibition of ATP and phosphate-dependent Ca2+ transport in subsequently prepared inside-out vesicles (IOV). Accumulation of phosphate into IOV in the presence of Ca2+ is virtually abolished by prior DIDS treatment, consistent with the presumed inhibition of the band III anion-exchange protein by this agent. No inhibition of Ca2+-activatable ATP hydrolysis is observed following DIDS treatment when open membranes are used to prevent development of ion gradients. This indicates that DIDS does not affect the inherent ATPase activity of the calcium pump (Waisman, D. M., Smallwood, J., Lafreniere, D., and Rasmussen, H. (1982) FEBS Lett. 145, 337-340). In IOV prepared from untreated cells, ATP-dependent Ca2+ uptake is stimulated by phosphate, sulfate, or chloride. Rates of Ca2+ uptake into DIDS-IOV are not increased by these anions. Lipid-permeable organic acids such as acetate, however, do promote Ca2+ transport in DIDS-IOV. Lipophilic anions incapable of transporting protons into the vesicle interior (nitrate and thiocyanate) support sustained uptake only when the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone is also added. These results support a model of the (Ca2+-Mg2+)-ATPase as a pump exchanging Ca2+ for protons, not transporting Ca2+ alone. Band III protein appears to promote Ca2+ transport in the presence of phosphate, sulfate, or chloride by exchanging external anion for the accumulating OH- (or HCO3-) produced by the calcium pump.     

Asymmetric inhibition of spicule formation in sea urchin embryos with low concentrations of gadolinium ion

As gastrulation proceeds during sea urchin embryogenesis, primary mesenchyme cells (PMCs) fuse to form syncytial cables, within which calcium is deposited as CaCO₃, and a pair of spicules is formed. Earlier studies suggested that calcium, previously sequestered by primary mesenchyme cells, is secreted and incorporated into growing spicules. We examined the effects of gadolinium ion (Gd³⁺), a Ca²⁺ channel blocker, on spicule formation. Gd³⁺ did not lead to a retardation of embryogenesis prior to the initiation of gastrulation and did not inhibit the ingression of PMCs from the blastula wall or their migration along the inner blastocoel surface. However, when embryos were raised in seawater containing submicromolar to a few micromolar Gd³⁺, of which levels are considered to be insufficient to block Ca²⁺ channels, a pair of triradiate spicules was formed asymmetrically. At 1–3 μmol/L Gd³⁺, many embryos formed only one spicule on either the left or right side, or embryos formed a very small second spicule. Induction of the spicule abnormality required the presence of Gd³⁺ specifically during late blastula stage prior to spicule formation. An accumulation or adsorption of Gd³⁺ was not detected anywhere in the embryos by X‐ray microanalysis, which suggests that Ca²⁺ channels were not inhibited. These results suggest that Gd³⁺ exerts an inhibitory effect on spicule formation through a mechanism that does not involve inhibition of Ca²⁺ channels.     

The isotopic effects of D2O in developing sea urchin eggs

When developing sea urchin eggs were treated with sea water containing 40% D2O (D2O-SW) at the 8-cell stage, the micromere formation was delayed and micromeres were larger than those seen in the control. But eggs returned to normal artificial sea water (NASW) at the 16- to 32-cell stage did not form abnormal spicules in larvae of Pseudocentrotus depressus. Little effect on the spicule formation of Hemicentrotus pulcherrimus was also noted. If the culture period in D2O-SW was extended until the hatching stage, the number of plutei with abnormal spicules increased. Primary mesenchyme cells of Pseudocentrotus depressus larvae failed to make two aggregated spicule rudiments on the ventral side of the larva and developed a ring-like spicule. This ring-like spicule, however, only occasionally occurred in the larvae of Hemicentrotus pulcherrimus. The cell cycle was longer in the presence of D2O. However, blastomeres managed to divide throughout the development. Larvae reared in 20% D2O-SW after the hatching stage developed into quasi-normal plutei but smaller than control. We found no exogastrulation in these larvae. Exogastrulation was found only in larvae continuously cultured in 40% D2O-SW from the early development. These results are inconsistent with previous reports made by other authors.     

A smooth muscle cell line suitable for the study of voltage sensitive calcium channels

A cell line originating from the fetal rat aorta has been studied with respect to 45Ca2+ uptake. Kinetic experiments showed an initial rapid uptake followed by a slow linear phase; both the initial rate and the maximum uptake were increased in the presence of 55 mM potassium chloride. The calcium channel antagonists, darodipine (PY 108-068) and verapamil, inhibited both the basal and the potassium chloride stimulated uptake. Neither tetrodotoxin nor furosemide affected either basal or depolarisation induced 45Ca2+ uptake. Blockade of the Na+/K+ ATPase by ouabain and of the Ca2+ ATPase by vanadate caused a net increase in cellular 45Ca2+ accumulation.     

Skeletogenesis in sea urchin interordinal hybrid embryos

Reciprocal interordinal crosses were made between the sea urchins Strongylocentrotus purpuratus and Lytechinus pictus. Previous research indicated that the expression of many L. pictus genes is reduced in the hybrid embryos. The S. purpuratus gene encoding the spicule matrix protein SM50 and the L. pictus gene encoding its orthologue LSM34 were both expressed at normal levels per gene copy in hybrid embryos, and in about 32 skeletogenic primary mesenchyme cells (PMCs) in hybrid and natural gastrulae. In many embryos of all crosses, 16 PMCs initially ingressed, while 32-64 PMCs were present in gastrulae. The skeletal spicules of most hybrid plutei were predominantly like those of S. purpuratus, consistent with the predominance of expression of S. purpuratus genes in hybrid embryos. The spicules of some hybrid plutei showed features characteristic of L. pictus, such as recurrent rods, branched body rod tips, or convergent ventral transverse rods; a few hybrid spicules were predominantly like those of L. pictus. Based on our observations and the literature, we propose the following. Cues from the ectodermal epithelium position the PMCs as they elaborate the initial triradiate spicules. Their orientation and outgrowth appears to be responsible for the convergence of the tips of body rods in most S. purpuratus and hybrid embryos, unlike in most L. pictus embryos. Variations among hybrid and natural embryos in skeletal branching pattern reflect differences in interpretation by PMCs of patterning cues produced by the ectodermal epithelium that probably have similar spatial distributions in the two species.     

Relation between the passive transport of calcium into vesicles of the myocardial sarcolemma and membrane potential

The effect of membrane potential on the passive 45Ca2+ uptake by cardial sarcolemmal vesicles was investigated. Membrane potentials were generated by the K+ gradient in the presence of valinomycin and were measured using fluorescent dye diS-C3-(5). It was shown that the 45Ca2+ influx into vesicles increased twice after membrane depolarization. Evaluation of the 45Ca2+ influx over a wide range of membrane potentials produced a profile similar to that of current-voltage relationships for single calcium channels in isolated cardiomyocytes. Passive 45Ca2+ transport was inhibited by 1 mM Cd2+ and Co2+. It is suggested that the voltage-dependent Ca2+ influx into vesicles occurs through Ca2+-channels.     

Aluminum Inhibits the Fast Phase of Voltage-Dependent Calcium Influx into Synaptosomes

Aluminum has been shown to have neurotoxic effects, but the mechanisms by which it acts are not well understood. Because it has been reported that aluminum can interact with Ca2+-binding sites, the possibility that aluminum might interfere with Ca2+ influx into synaptosomes was examined. At concentrations of 50 microM and greater, aluminum significantly inhibited the fast phase (0-1 s) of the voltage-dependent uptake of 45Ca2+ into synaptosomes. Higher concentrations of aluminum also reduced 45Ca2+ uptake measured at 1 s in nondepolarizing media and inhibited the slow phase of 45Ca2+ uptake into synaptosomes whether they were suspended in either low K or high K media. The possibility that aluminum competitively inhibits the fast phase of Ca2+ influx was investigated. Aluminum (250 microM) increased the apparent KT (concentration of Ca2+ at which Ca2+ transport is half maximal) for 45Ca2+ of fast phase voltage-dependent channels and slightly decreased the maximal influx (Jmax). These effects are characteristic of a mixed type inhibitor, and the apparent Ki for Al3+ is estimated to be 0.64 mM. The interaction of aluminum with the fast phase of voltage-dependent calcium influx may disrupt intraneuronal calcium homeostasis and may also represent a means by which aluminum could accumulate intraneuronally.     

A calcium-binding, asparagine-linked oligosaccharide is involved in skeleton formation in the sea urchin embryo

We have previously identified a 130-kD cell surface protein that is involved in calcium uptake and skeleton formation by gastrula stage embryos of the sea urchin Strongylocentrotus purpuratus (Carson et al., 1985. Cell. 41:639-648). A monoclonal antibody designated mAb 1223 specifically recognizes the 130-kD protein and inhibits Ca+2 uptake and growth of the CaCO3 spicules produced by embryonic primary mesenchyme cells cultured in vitro. In this report, we demonstrate that the epitope recognized by mAb 1223 is located on an anionic, asparagine-linked oligosaccharide chain on the 130-kD protein. Combined enzymatic and chemical treatments indicate that the 1223 oligosaccharide contains fucose and sialic acid that is likely to be O-acetylated. Moreover, we show that the oligosaccharide chain containing the 1223 epitope specifically binds divalent cations, including Ca+2. We propose that one function of this negatively charged oligosaccharide moiety on the surfaces of primary mesenchyme cells is to facilitate binding and sequestration of Ca+2 ions from the blastocoelic fluid before internalization and subsequent deposition into the growing CaCO3 skeleton.     

Probable Role of Allylisothiocyanate-Sensitive H+, K+-ATPase in Spicule Calcification in Embryos of the Sea Urchin, Hemicentrotus pulcherrimus

In embryos of the sea urchin, Hemicentrotus pulcherrimus , as well as in cultured cells derived from isolated micromeres, spicule formation was inhibited by allylisothiocyanate, an inhibitor of H⁺, K⁺-ATPase, at above 0.5 μM and was almost completely blocked at above 10 μM. Amiloride, an inhibitor of Na⁺, H⁺ antiporter, at above 100 μM exerted only slight inhibitory effect, if any, on spicule formation. Intravesicular acidification, determined using [ dimethylamine -¹⁴C]-aminopyrine as a pH probe, was observed in the presence of ATP and 200 mM KCl in microsome fraction obtained from embryos at the post gastrula stage, at which embryos underwent spicule calcification. Intravesicular acidification and K⁺-dependent ATPase activity were almost completely inhibited by allylisothiocyanate at 10 μM. Allylisothiocyanate-sensitive ATPase activity was found mainly in the mesenchyme cells with spicules isolated from prisms. H⁺, K⁺-ATPase, an H⁺ pump, probably mediates H⁺ release to accelerate CaCO₃ deposition from Ca²⁺, CO₂ and H₂O in the primary mesenchyme cells. Intravesicular acidification was stimulated by valinomycin at the late gastrula and the prism stages but not at the pluteus stage. K⁺ permeability probably increases after the prism stage to activate H⁺ release.     

Beta-adrenergic stimulation evokes a rapid, Ca2+-dependent stimulation of endocytosis, hexose and amino acid transport associated with increased Ca2+ fluxes in mouse kidney cortex

The beta-adrenergic agonist 1-isoproterenol evokes an acute (less than 5 min) stimulation of endocytosis, hexose transport and amino acid transport, measured by the temperature-sensitive uptake of HRP, 3H-DG and 14C-AIB, in mouse kidney cortex slices. This stimulation is concentration dependent and is maximal at 10(-8)-10(-7) M isoproterenol. Peroxidase cytochemistry showed that the hormonal increase in HRP uptake is confined to proximal tubules. The rapid membrane response is abolished in a calcium-free medium and by the beta-adrenergic antagonist propranolol, indicating Ca2+- and beta-adrenoreceptor-dependence. Isoproterenol (1 microM) rapidly (less than 30 sec) stimulates the influx and efflux of 45Ca in cortex slices. Isoproterenol also decreased mitochondrial 45Ca and increased soluble 45Ca. These results indicate that beta-adrenergic stimulation of membrane transport functions involves an increased influx of extracellular calcium and a mobilization of intracellular (mitochondrial) calcium. An increase in cytosolic Ca2+ concentration appears to be the regulatory signal for these membrane transport processes.     

Enhancement of spicule formation and calcium uptake by monoclonal antibodies to fibronectin-binding acid polysaccharide in cultured sea urchin embryonic cells

The effect of monoclonal antibodies to fibronectin-binding acid polysaccharide (anti-FAPS) on differentiation of primary mesenchyme cells and spicule formation was examined in cultured embryonic cells isolated from the sea urchin, Hemicentrotus pulcherrimus. Spicule formation of micromere-derived cells was enhanced by anti-FAPS. The increase of spicule formation correlated with the increase of calcium uptake into micromere-derived cells and spicules. Furthermore, both spicule formation and calcium uptake were inhibited by calcium-channel blockers (verapamil, diltiazem and nicardipine) and divalent ions (manganese and cobalt). These results suggest that FAPS, a component of the blastocoelic extracellular matrix surrounding the primary mesenchyme cells, may regulate the level of calcium uptake and spicule formation.     

Physiological characterization of 45Ca2+ and 65Zn2+ transport by lobster hepatopancreatic endoplasmic reticulum

The crustacean hepatopancreas is an epithelial-lined, multifunctional organ that, among other activities, regulates the flow of calcium into and out of the animal's body throughout the life cycle. Transepithelial calcium flow across this epithelial cell layer occurs by the combination of calcium channels and cation exchangers at the apical pole of the cell and by an ATP-dependent, calcium ATPase in conjunction with a calcium channel and an Na+/Ca2+ antiporter in the basolateral cell region. The roles of intracellular organelles such as mitochondria, lysosomes, and endoplasmic reticulum (ER) in transepithelial calcium transport or in transient calcium sequestration are unclear, but may be involved in transferring cytosolic calcium from one cell pole to the other. The ER membrane has a complement of ATP-dependent calcium ATPases (SERCA) and calcium channels that regulate the uptake and possible transfer of calcium through this organelle during periods of intense calcium fluxes across the epithelium as a whole. This investigation characterized the mechanisms of calcium transport by lobster hepatopancreatic ER vesicles and the effects of drugs and heavy metals on them. Kinetic constants for 45Ca2+ influx under control conditions were K(n) (m)=10.38+/-1.01 microM, J(max)=14.75+/-1.27 pmol/mg protein x sec, and n=2.53+/-0.46. The Hill coefficient for 45Ca2+ influx under control conditions, approximating 2, suggests that approximately two calcium ions were transported for each transport cycle in the absence of ATP or the inhibitors. Addition of 1 mM ATP to the incubation medium significantly (P<0.01) elevated the rate of 45Ca2+ influx at all calcium activities used and retained the sigmoidal nature of the transport relationship. The kinetic constants for 45Ca2+ influx in the presence of 1 mM ATP were K(n) (m)=12.76+/-0.91 microM, J(max)=25.46+/-1.45 pmol/mg protein x sec, and n=1.95+/-0.15. Kinetic analyses of ER 65Zn2+ influx resulted in a sigmoidal relationship between transport rate and zinc activity under control conditions (K(n) (m)=38.63+/-0.52 microM, J(max)=19.35+/-0.17 pmol/mg protein x sec, n=1.81+/-0.03). The Addition of 1 mM ATP enhanced 65Zn2+ influx at each zinc activity, but maintained the overall sigmoidal nature of the kinetic relationship. The kinetic constants for zinc influx in the presence of 1 mM ATP were K(n) (m)=34.59+/-2.31 microM, J(max)=26.09+/-1.17 pmol/mg protein x sec, and n=1.96+/-0.17. Both sigmoidal and ATP-dependent calcium and zinc influxes by ER vesicles were reduced in the presence of thapsigargin and vanadate. This investigation found that lobster hepatopancreatic ER exhibited a thapsigargin- and vanadate-inhibited, SERCA-like, calcium ATPase. This transporter displayed cooperative calcium transport kinetics (Hill coefficient, n approximately 2.0) and was inhibited by the heavy metals zinc and copper, suggesting that the metals may reduce the binding and transport of calcium when they are present in the cytosol.     

Involvement of anion channel in signal transduction of early defense responses elicited by harpinPss in tobacco]

No matter when anion channel inhibitors, DIDS (4, 4'-diisothiocyanatostilbene-2, 2'-disulfonic acid) and A9C (anthracene-9-carboxylic acid) added (before, at the same time of or after harpinPss treatment), they can inhibit harpinPss-induced hypersensitive response in tobacco seedlings and release of active oxygen and extracellular alkalinization in tobacco suspension cells. DIDS and A9C also inhibit harpinPss-induced Ca2+ influx. In all these cases, DIDS is more efficient than A9C. It is postulated that anion channel positively regulates calcium channel in plasma membrane, and harpinPss may function through signal transduction mediated by anion channel and calcium channel to regulate cellular Ca2+ concentration and defense responses.     

Proteins of calcified endoskeleton: II partial amino acid sequences of endoskeletal proteins and the characterization of proteinaceous organic matrix of spicules from the alcyonarian, Synularia polydactyla

Calcified organic substances in the skeleton contain a protein-polysaccharide complex taking a key role in the regulation of bio-calcification. However, information concerning the matrix proteins in alcyonarian and their effect on calcification process is still unknown. For this reason, we have studied the organic matrix of endoskeletal spicules from the alcyonarian coral, Synularia polydactyla, to analyze the proteins with their sequences and investigate the functional properties by a molecular approach. The separated spicules from the colony were identified by scanning electron microscope (SEM). The soluble organic matrix comprised 0.04% of spicule weight. By recording decline of pH in the experimental design, the inhibitory effect of the matrix on CaCO3 precipitation was revealed. Prior to electrophoresis, our analysis of proteins extracted from the soluble organic matrix of the spicules revealed an abundance of proteins in molecular weight. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the preparations showed seven bands of proteins with an apparent molecular mass of 109, 83, 70, 63, 41, 30 and 22 kDa. The proteins were electrophoresed on Tricine-SDS-PAGE after electro-elution treatment, and then transferred to polyvinylidene difluoride (PVDF) membranes and their N-termini were sequenced. Two bands of proteins of about 70 and 63 kDa successfully underwent N-terminal amino acid sequencing. For the detection of calcium binding proteins, a Ca2+ overlay analysis was conducted on the extract by 45Ca autoradiography. The 109 and 63 kDa calcium binding proteins were found to be radioactive. Periodic acid schiff staining indicated that 83 and 63 kDa proteins were glycosylated. An assay for carbonic anhydrase, which is thought to play an important role in the process of calcification revealed low level of the activity. These findings suggest that the endoskeletal spicules of alcyonarian corals have protein-rich organic matrices, which might be related to the calcification process.