Calcium-dependent protein kinase from apple fruit membranes is calmodulin-independent but has calmodulin-like properties

Crude Ca²⁺-activated protein kinase from membranes of apple (Malus domestica L. Borkh., Cox's Orange Pippin) fruit can be partially purified to yield a Ca²⁺-dependent protein kinase whose activity is apparently not regulated by calmodulin. The autophosphorylating catalytic subunit of this protein kinase shows a Ca²⁺-dependent mobility shift of approx. 10 kilodaltons (kDa) on sodium dodecyl sulphate-polyacrylamide gel electrophoresis; in the absence of added Ca²⁺ or ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid (EGTA) its apparent molecular mass is approx. 50 kDa. The Ca²⁺-dependent protein kinase is inhibited by the calmodulin antagonists N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide and trifluoperazine with IC₅₀ values of approx. 45 M and 15 M, respectively. These similarities between the protein kinase and calmodulin indicate that the kinase may be a calmodulin-like protein.Abbreviations DEAE diethylaminoethyl - EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - Hepes 4-(-2-hydroxyethyl)-1-piperazineethanesulphonic acid - kDa kilodalton - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulphate - W7 N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide - W5 N-(6-aminohexyl)-naphthalenesulphonamide     

Identification of calmodulin in the green alga Mougeotia and its possible function in chloroplast reorientational movement

A soluble protein was isolated from Mougeotia by chloropromazine-sepharose 4 B affinity chromatography. The protein matches the properties of calmodulin in terms of heat stability, Ca²⁺-dependent electrophoretic mobility in sodium-dodecyl-sulfate polyacrylamide gels, and its ability to activate cyclic nucleotide phosphodiesterase in a Ca²⁺-dependent manner. Phytochrome-mediated chloroplast reorientational movement in Mougeotia was inhibited by the calmodulin antagonist trifluoperazine, a hydrophobic compound, or N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a hydrophilic compound; 50% inhibition (IC₅₀) of chloroplast movement is caused by 20–50 mol l^-1 trifluoperazine or 100 mol l^-1 W-7. The Ca²⁺-calmodulin may act as an intermediate in the chloroplast reorientational response in Mougeotia governed by phytochrome.Abbreviations EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - SDS sodium dodecyl sulfate - W-7 N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide     

Calcium-regulated appressorium formation of the entomopathogenic fungusZoophthora radicans

Summary The fungusZoophthora radicans (Zygomycetes: Entomophthorales) requires external Ca²⁺ for appressorium formation but not for conidial germination. The number of appressoria formed depends on the Ca²⁺ concentration of the medium. At low [Ca²⁺] (100 pM) nuclear division and germ tube growth are significantly reduced compared to higher Ca²⁺ concentrations (10 and 1,000 M). By contrast, neither external K⁺ nor external Cl^– is needed for germination or appressorium formation. Treatment of conidia with a Ca²⁺-antagonist, Nd³⁺, and a Ca²⁺-channel blocker, nifedipine, inhibits appressorium formation, showing that a Ca²⁺ influx is required for appressorium formation. Furthermore, the partial yet saturating inhibition by nifedipine and complete inhibition by Nd³⁺ indicates that at least two kinds of Ca²⁺ channels are involved in appressorium formation. A contribution of intracellular Ca²⁺ to the signal transduction chain for the formation of appressoria is demonstrated by the inhibitory effect of the intracellular Ca²⁺ antagonist TMB-8. The calmodulin antagonists R24571, TFP, W-7, and W-5 inhibit appressorium formation at concentrations which have no effect on germination. The data presented in this paper are consistent with the hypothesis that a Ca²⁺/calmodulin system is involved in regulating appressorium formation. However, since the direct effects of the drugs were not specifically tested on their proposed binding sites, we leave room for alternative hypotheses that have yet to be formulated.Abbreviations A-9-C 9-anthracenecarboxylic acid - DAPI 4,6 diamino-2-phenylindole - EGTA ethylene glycol bis(-aminoethylether)-N,N-tetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - H-7 N-(2-methylamino)ethyl-5-isoquino-linesulphonamide dihydrochloride - IC₅₀ concentration of inhibitor that causes 50% inhibiton - R24571 (calmidazolium) 1-[bis-(4-chlorophenyl)methyl]-3-[2,4-dichloro--(2,4-dichlorobenzyloxy)phenethyl]-imidazolium chloride - TEA tetraethylammonium - TFP (trifluoperazine) 10-[3-(4-methylpiperazine-1-yl)-propyl]-2-trifluomethylphenothiazine - TMB-8 8-(diethylamino)octyl 3,4,5-trimethoxybenzoate hydrochloride - W-5 N-(6-aminohexyl)-1-naphthalene-sulfonamide - W-7 N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide     

Rapid Ca2+ extrusion via the Na+/Ca2+ exchanger of the human platelet

Summary This communication reports the kinetics of the Na⁺/ Ca²⁺ exchanger and of the plasma membrane (PM) Ca²⁺ pump of the intact human platelet. The kinetic properties of these two systems were deduced by studying the rate of Ca²⁺ extrusion and its Na⁺ dependence for concentrations of cytoplasmic free Ca²⁺ ([Ca²⁺]_cyt) in the 1–10-m range. The PM Ca²⁺ATPase was previously characterized (Johansson, J.S. Haynes, D.H. 1988. J. Membrane Biol. 104:147–163) for [Ca²⁺]_cyt] 1.5 m with the fluorescent Ca²⁺ indicator quin2 (K _d= 115 nm). That study determined that the PM Ca²⁺ pump in the basal state has a V _max = 0.098 mm/min, a K _m= 80 nm and a Hill coefficient = 1.7. The present study extends the measurable range of [Ca²⁺]_cyt with the intracellular Ca²⁺ probe, rhod2 (K _d= 500 nm), which has almost a fivefold lower affinity for Ca²⁺. An Appendix also describes the Mg²⁺ and pH dependence of the K _dand fluorescence characteristics of the commercially available dye, which is a mixture of two molecules. Rates of active Ca²⁺ extrusion were determined by two independent methods which gave good agreement: (i) by measuring Ca²⁺ extrusion into a Ca²⁺-free medium (above citation) or (ii) by the newly developed ionomycin short-circuit method, which determines the ionomycin concentration necessary to short circuit the PM Ca²⁺ extrusion systems. Absolute rates of extrusion were determined by knowledge of how many Ca²⁺ ions are moved by ionomycin per minute. The major findings are as follows: (i) The exchanger is saturable with respect to Ca²⁺ with a K _m= 0.97 ± 0.31 m and V_max = 1.0 ± 0.6 mm/ min. (ii) At high [Ca²⁺]_cyt, the exchanger works at a rate 10 times as large as the basal V _max of the PM Ca²⁺ extrusion pump. (iii) The exchanger can work in reverse after Na⁺ loading of the cytoplasm by monensin. (iv) The PM Ca²⁺ extrusion pump is activated by exposure to [Ca²⁺]_cyt 1.5 m for 20–50 sec. Activation raises the pump V _max to 1.6 ± 0.6 mm/min and the K _mto 0.55 ± 0.24 m. (v) The Ca²⁺ buffering capacity of the cytoplasm is 3.6 mm in the 0.1 to 3 m range of [Ca²⁺]_cyt. In summary, the results show that the human platelet can extrude Ca²⁺ very rapidly at high [Ca²⁺]_cyt. Both the Na⁺/Ca²⁺ exchanger and Ca²⁺ pump activation may prevent inappropriate platelet activation by marginal stimuli.Abbreviations cAMP cyclic adenosine 3,5-monophosphate - cGMP cyclic guanosine 3,5,-monophosphate - Ca-CAM calcium calmodulin; - DT dense tubules - B intrinsic cytoplasmic Ca²⁺ binding sites - R rhod2 or 5-(3,6-bis(dimethylamino)xanth-9-yl)-1-(2-amino-4-hy droxy lphenoxy)-2-(2-amino-5-methylphen- oxy)ethane-N,N,NN-tetraacetic acid - [Ca²⁺]_cyt cytoplasmic Ca²⁺ activity - quin2 2-[[2-bis[(carboxymethyl)amino]-5-methyl-phenoxy]methyl]-6-methoxy-8-[bis(carboxymethyl)amino]quinoline - V or V_extrusion true rate of Ca²⁺ extrusion - fura-2 1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy]-2-(2-amino-5-methylphenoxy)-ethane-N,N,NN-tetraacetic acid - AM acetoxymethyl ester - DMSO dimethylsulfoxide - CTC chlortetracycline - EGTA ethyleneglycol-bis(-aminoethyl ether) N,N,N,N- tetraacetic acid - HEPES 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid - NMDG N-methyl-d-glucamine - PIPES 1,4-piperazine-bis-(ethanesulfonic acid) - HPLC high performance liquid chromatography - _I fraction of high-affinity rhod2 complexed with Ca²⁺ - F the observed fluorescence - F_min the minimal fluorescence observed in the absence of Ca²⁺ - F_max the maximal fluorescence observed when the dye is saturated with Ca²⁺ - X₁ the fraction of high-affinity dye - K _d,1 dissociation constant of high-affinity dye - K _d,2 dissociation constant of the low-affinity dye - -d₁/dt rate of Ca²⁺ removal from the rhod2-Ca complex; - -dF/dt the slope representing the absolute rate of fluorescence decrease in a progress curve - F_max (F_max — F_min)_cyt difference between maximal and minimal fluorescence for cytoplasmic high affinity form of rhod2 - F₅₀ fluorescence of the high-affinity form ofrhod2for[Ca²⁺]_cyt=50 nM - [Ca²⁺]₀ external Ca²⁺concentration - K _p proportionality constant between the total number of Ca²⁺ ions moved and the change in high-affinity rhod2 complexation to Ca² - (d[Ca²⁺]_{cyt, T})/dt rate of Ca²⁺ influx obtained with maximal levels of ionomycin - k_leak rate constant for passive inward Ca²⁺ leakage - k_inno rate constant for ionomycin-mediated Ca²⁺ influx - T total - [rhod2]_cyt,T total intracellular rhod2 concentration - [quin2]_cyt,T total intracellular quin2 concentration - [B]_T total cytoplasmic buffering capacity - A[Ca²⁺]_cyt,T total number of Ca²⁺ ions moved into the cytoplasm - [rhod2-Ca]_{cyt, T} change in concentration of total intracellular high-affinity rhod2 complexed to Ca²⁺ - [B-Ca]_T change in concentration of total cytoplasmic binding sites complexed to Ca²⁺ - [quin2]_{cyt, T} change in concentration of total intracellular quinl complexed to Ca²⁺ - change in the degree of intracellular quin2 saturation - ₁ change in degree of saturation of cytoplasmic high-affinity rhod2 - ₁-/t rate of change in degree of saturation of cytoplasmic high affinityrhod2 - V_obs observed rate of Ca²⁺ removal from the rhod2-Ca complex - V_{8.3 m} the rate of Ca²⁺ removal from the high affinity rhod2-Ca complex at [Ca²⁺]_cyt = 8.3 m - /t rate of change in of the degree of quin2 saturation - [Ca²⁺]_cytT/_t initial linear rate of ionomycin-mediated Ca²⁺ influx - EC₅₀ effective concentration giving a half-maximal effect - [Na⁺]_cyt cytoplasmic Na⁺ activity - CAM calmodulin - ACN acetonitrile - TFA trifuloroacetic acid     

A non-specific Ca2+ (or Mg2+)-stimulated ATPase in rat heart sarcoplasmic reticulum

ATPase activity in rat heart sarcoplasmic reticulum was stimulated in a concentration-dependent manner by both Ca²⁺ and Mg²⁺ in the complete absence of the other cation. Increasing concentrations of Mg²⁺ produced an apparent inhibition of the Ca²⁺-dependent ATP hydrolysis. CDTA (trans-1,2-diaminocyclohexane-N,N,N,N-tetraacetate) had no effect on these responses. The results indicate the presence of a low affinity non-specific divalent cation-stimulated ATPase in rat heart sarcoplasmic reticulum. However, sarcoplasmic reticulum vesicles transported Ca²⁺ with a high affinity (K_0.5 Ca²⁺ = 0.41 M) suggesting the presence of a high affinity Ca²⁺-transporting ATPase. Calmodulin did not stimulate rat heart sarcoplasmic reticulum ATPase activity over a range of Ca²⁺ and Mg²⁺ concentrations and failed to stimulate membrane phosphorylation and Ca²⁺ transport into sarcoplasmic reticulum vesicles. Calmodulin antagonists trifluoperazine and compound 48180 did not affect the ATPase activity. Catalytic subunit of cAMP-dependent protein kinase was also ineffective in stimulating the ATPase activity. These results suggest the presence of an ATPase activity in rat heart sarcoplasmic reticulum with different properties from the high affinity Ca²⁺-pumping ATPase previously characterized in dog heart and other species.Abbreviations cAMP adenosine 3,5-monophosphate - CaM calmodulin - CDTA trans-1,2-diaminocyclohexane-N,N,N,N-tetraacetate - EDTA ethylene-diaminetetraacetate - EGTA ethylene glycol bis(-aminoethyl ether)-N,N,N,N-tetraacetate - PLB phospholamban - SR sarcoplasmic reticulum - TFP trifluoperazine     

Activation of chitin synthetase from Phycomyces blakesleeanus by calcium and calmodulin

Levels of basal chitin synthetase in cell-free extracts from Phycomyces blakesleeanus were reduced by breakage of cells in the presence of EDTA or EGTA. Addition of Ca²⁺ to these extracts activated chitin synthetase. Maximal activation was obtained after 2 h at a Ca²⁺ concentration of 2–5 mM. Activation by calcium was not reduced by any protease inhibitor tested but benzamidine, whereas the weak proteolytic activity of the extracts was inhibited by antipain. Larger levels of chitin synthetase activation were obtained by the simultaneous addition of calcium and calmodulin in most, but not all extracts. This further activation by calmodulin was prevented by TFP. ATP or cAMP did not stimulate activation by calcium or calcium-calmodulin.Abbreviations EGTA ethylene glycol-bis(B-aminoethylether)-N,NN-tetraacetic acid - GlcNAc N-acetyl-d-glucosamine - PMSF phenylmethylsulfonyl fluoride - SBTI soybean trypsin inhibitor - TFP trifluoperazine - TLCK N-p-tosyl-l-lysine choromethyl ketone - UDPGlcNAc uridine diphosphate N-acetyl-d-glucosamine     

Calmodulin regulates the Ca2+-dependent slow-vacuolar ion channel in the tonoplast of Chenopodium rubrum suspension cells

The patch-clamp technique was applied to vacuoles isolated from a photoautotrophic suspension cell culture of Chenopodium rubrum L. and vacuolar clamp currents, which are predominantly carried by the previously identified Ca²⁺-dependent slow vacuolar (SV) ion channels, were recorded. These currents, which were activated by 1-s voltage pulses of -100 mV (vacuolar interior negative) in the presence of 100 M Ca²⁺ (cytosolic side), could be blocked completely and reversibly by the calmodulin antagonist W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide] and its chlorine-deficient analogue W-5; half-maximum inhibition was found at approx. 6 M for W-7 and 70 M for W-5. Inhibition was reversed by addition of 1 g · ml^–1 calmodulin purified from Chenopodium cell suspensions; reversal by bovine brain calmodulin was scarcely appreciable. We conclude that cytosolic calmodulin mediates the Ca²⁺ dependence of the SV-channel in the Chenopodium tonoplast.Abbreviations SV-channel slowly activated, vacuolar ion channel - W-5 N-(6-aminohexyl)-1-naphthalenesulfonamide - W-7 N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide We acknowledge support by the Deutsche Forschungsgemeinschaft and the Bundesminister für Forschung und Technologie, Bonn, and by the Justus-Liebig-Universität Giessen (to W.B.)     

Wound-healing motility in the green alga Ernodesmis: calcium ions and metabolic energy are required

Wounding a giant cell of the marine alga Ernodesmis verticillata (Kützing) Børgesen (Chlorophyta) induces two concomitant motility phenomena: longitudinal contraction of the protoplasm away from the wound site, and centripetal contraction of the cut end around the central vacuole. Healing is complete within 30 min of wounding. Mechanical extrusion of the protoplasm into the medium with a teasing needle is followed by contraction of the protoplasm into numerous spherical protoplasts within 60 min. Utilizing a simple defined medium, it is shown that motility is almost completely inhibited by the absence of exogenous free Ca²⁺, with 5.0 mM ethylene glycol bis-(-aminoethyl ether)-N,N,N,N-tetraacetic acid present. This inhibition is reversible by rinsing the cells with Ca²⁺-containing medium. Similarly, extruded cytoplasm fails to exhibit motility in Ca²⁺-free medium. The threshold concentration of exogenous free Ca²⁺ is approx. 10^-7 M for wound-induced contraction. The ions Ba²⁺, Cd²⁺ and Sr²⁺ will substitute for Ca²⁺, but the rate of contraction is one-half that with Ca²⁺ present. Although darkness has no inhibitory effect, lower temperature (15°C), cyanide, or micromolar amounts of phosphorylation uncouplers reversibly slow protoplasmic motility in wounded cells and extruded cytoplasm. Carbonylcyanide m-chlorophenylhydrazone and carbonylcyanide p-trifluoromethoxyphenylhydrazone are especially potent inhibitors. These results indicate that cellular wound healing utilizes metabolic energy and requires exogenous free Ca²⁺, implying that motility in Ernodesmis is a true contractile process. Since 1.0 mM La³⁺ completely and reversibly prevents contraction, it is postulated that Ca²⁺ fluxes may actually trigger motility.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - DMSO dimethylsulfoxide - DNP 2,4-dinitrophenol - EGTA ethylene glycol bis-(-aminoethyl ether)-N,N,N,N-tetraacetic acid - FCCP carbonylcyanide p-trifluoromethoxyphenylhydrazone     

Ca2+-induced fragmentation of actin filaments in pollen tubes

Summary To control the intracellular free Ca²⁺ concentration from the cell exterior, pollen tubes ofLilium longiflorum were treated with a Ca²⁺ ionophore, A23187. Cytoplasmic streaming was inhibited when the free Ca²⁺ concentration of the external medium ([Ca²⁺]) was raised to 5×10^–6 M or higher. At [Ca²⁺] below 1×10^–6 M, the rhodamine-phalloidin stained actin filaments appeared straight and thin. However, at [Ca²⁺] which inhibited cytoplasmic streaming, the actin filaments appeared fragmented. In pollen tubes, Ca²⁺ regulation of cytoplasmic streaming may be linked not only to myosin (Shimmen 1987) but also to actin.Abbreviations ATP adenosine-5-triphosphoric acid - [Ca²⁺] concentration of free Ca²⁺ - EGTA ethyleneglycol-bis-(-aminoethylether)N,N,N,N-tetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - PIPES piperazine-N,N-bis(2-ethanesulfonic acid) - Rh-ph rhodamine-conjugated phalloidin     

Effect of calcium,its inhibitors,and heavy metals on the growth cycle of peanut cell aggregates

During the normal growth cycle of peanut (Arachis hypogaea L.) cells, cultured in suspension medium, cell aggregates of <0.5 mm were formed during the log phase and grew to aggregates of >0.5 mm during late growth phase. Calmodulin rose to its original level during <0.5 mm aggregate formation following an initial 50% drop. Observations by UV microscopy showed that calmodulin. Ca²⁺ was centered in intense fluorescent sites. Calmodulin antagonists and a calcium chelator inhibited <0.5 mm aggregate formation as well as protein accumulation. The chelator suppressed cationic peroxidase isozyme release, while the antagonists had some partial effect on the anionic isozyme. Some heavy metals such as cadmium, mercury, lead and cobalt at low concentrations would allow continued growth of >0.5 but not of the <0.5 populations. At high (1 mM) concentrations these ions caused arrested growth. At low (10 M) levels and in the presence of 3 mM calcium they had a synergistic effect.Abbreviations AOA amino oxy acetic acid - BCA big cell aggregates - CPZ chlorpromazine - EGTA ethylene glycol bis (-aminoethyl ether) - N, N, N, N tetra acetic acid - SCA small cell aggregates - TFP trifluoperazine     

Some properties of adenosine 3′,5′-cyclic monophosphate phosphodiesterase in the superior cervical ganglion of the guinea pig

Adenosine 3,5-cyclic monophosphate (cAMP) phosphodiesterase activity in crude guinea-pig superior cervical ganglion homogenates was assayed under a variety of experimental conditions. Two forms of cAMP phosphodiesterase were found, one with high and the other with low affinity for the substrate. TheK _m values were about 1 and 110 M respectively. Imidazole slightly but constantly stimulated the former enzyme form over a wide range of concentrations and l-methyl-3-isobutylxanthine was a weak competitive inhibitor with aK _i value of 90 M. Low affinity cAMP phosphodiesterase activity was increased by calmodulin and Ca²⁺. This stimulation was not observed in the presence of trifluoperazine, a specific inhibitor of calmodulin. On the other hand, neither [d-Ala²]met-enkephalinamide nor prostaglandin E₂, alone or in combination, influenced high affinity cAMP phosphodiesterase.     

Inhibitory ca2+-control of movement of beads coated withphysarum myosin along actin-cables inChara internodal cells

Summary The actin-activated ATPase activityPhysarum myosin was shown to be inhibited of M levels of Ca²⁺. To determine if Ca²⁺ regulates ATP-dependent movement ofPhysarum myosin on actin, latex beads coated withPhysarum myosin were introduced intoChara cells by intracellular perfusion. In perfusion solution containing EGTA, the beads moved along the parallel arrays ofChara actin filaments at a rate of 1.0–1.8 m/sec; however, in perfusion solution containing Ca²⁺, the rate reduced to 0.0–0.7 m/sec. The movement of beads coated with scallop myosin, whose actin-activated ATPase activity is activated by Ca²⁺, was observed only in the perfusion solution containing Ca²⁺, indicating that myosin is responsible for the inhibitory effect of Ca²⁺ onPhysarum myosin movement. The involvement of this myosin-linked regulation in the inhibitory effect of Ca²⁺ on the cytoplasmic streaming observed inChara internodal cell andPhysarum plasmodium was discussed.Abbreviations ATP adenosine 5-triphosphate - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - EGTA ethyleneglycolbis(-aminoethylether) N,N,N,N-tetraacetic acid - PIPES piperazine-N,N-bis(2-ethanesulfonic acid)     

Requirement for calcium ions in mycobacteriophage I3 DNA injection and propagation

Ca²⁺ ions are absolutely necessary for the propagation of mycobacteriophage I3 in synthetic medium. These ions are required for successful infection of the host and during the entire span of the intracellular development of the phage. A direct assay of the phage DNA injection using ³²[P] labelled phage, showns that Ca²⁺ ions are necessary for the injection process. The injection itself is a slow process and takes 15 min to complete at 37°C. The bacteria infected in presence of Ca²⁺ tend to abort if the ions are subsequently withdrawn from the growth medium. The effect of calcium withdrawal is maximally felt during the early part of the latent period; however, later supplementation of Ca²⁺ ions salvage phage production and the mature phage progeny appear after a delayed interval, proportional to the time of addition of Ca²⁺.Abbreviations moi multiplicity of infection - PFU plaque forming units - EGTA ethylene-glycol-bis (-aminoethyl ether) N,N-tetraacetic acid     

Stereo-isomer specific induction of renal cell apoptosis by synthetic muramyl dipeptide lpar;N-acetylmuramyl-L-alanyl-D-isoglutamine)

In order to understand the modification of -adrenoceptor linked signal transduction by changes in the intracellular Ca²⁺, we examined the status of -adrenoceptors (-ARs), G-proteins and adenylyl cyclase (AC) in Ca²⁺-deficiency and Ca²⁺-overload by perfusing the isolated rat heart with Ca²⁺-free medium for 5 min and Ca²⁺-containing medium for 5 min following Ca²⁺-free perfusion, respectively. Ca²⁺-depletion caused not only an increase in basal, isoproterenol-, Gpp(NH)p-, NaF- and forskolin-stimulated AC activities but also produced an increase in the ₁-AR affinity and density as well as up-regulation of G_s-protein function and uncoupling of G_i-protein to AC. Ca²⁺-repletion for 5 min following 5 min Ca²⁺-free perfusion reversed the increased AC activities to varying degrees. The ₁-AR affinity was further increased upon Ca²⁺-repletion whereas its density was decreased. Ca²⁺-repletion also decreased protein content for AC and -AR kinase but augmented the changes in G_s- and G_i-protein functions. Although low Na⁺- medium perfusion during Ca²⁺-depletion prevented the changes in G-proteins during both Ca²⁺-depletion and Ca²⁺-repletion periods, the increased ₁-AR affinity and density as well as changes in AC activities due to Ca²⁺-depletion were not affected while alterations due to Ca²⁺-repletion were fully prevented. These results suggest that changes in Ca²⁺-homeostasis may represent a mechanism for alterations in the -adrenergic signal transduction pathway in the heart under pathological conditions.     

Turgor regulation and cytoplasmic free Ca2+ in the algaLamprothamnium

Summary In internodal cells ofLamprothamnium succinctum, turgor regulation in response to hypotonie treatment is inhibited by lowering external Ca²⁺ concentration ([Ca²⁺]_e) from 3.9 (normal) to 0.01 (low) mM. In order to clarify whether a change in the cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_c) is involved in turgor regulation, the Ca²⁺ sensitive protein aequorin was injected into the cytoplasm of internodal cells. A large transient light emission was observed upon hypotonic treatment under normal [Ca²⁺]_e but not under low [Ca²⁺]_e. Thus hypotonic treatment induces a transient increase in [Ca²⁺]_c under normal [Ca²⁺]_e but not under low [Ca²⁺]_e.Abbreviations ASW artificial sea water - _i cellular osmotic pressure - [Ca²⁺]_c cytoplasmic free Ca²⁺ concentration - EDTA ethylenediamine-tetraacetic acid - EGTA ethylenglycol-bis(-aminoethyl ether(N,N-tetraacetic acid - [Ca²⁺]_e external Ca²⁺ concentration - _e external osmotic pressure - GM glass micropipette - GP glass plate - HEPES N-2-hydroxyethylpiperazine-N-2-ethansulfonic acid - MS microscope stage - OL objective lens - PIPES piperazine-N-N-bis(2-ethanesulfonic acid) - W Weight     

Regulation of exocytosis in electrically permeabilized insulin-secreting cells. Evidence for Ca2+ dependent and independent secretion

The regulation of insulin secretion from RINm5F cells exposed to high voltage discharge has been investigated. Electron microscopy revealed that the overall structure of the cells was preserved after permeabilization. In this preparation insulin release was stimulated by Ca²⁺ (EC₅₀=2.4 M). The stable GTP analogue GTPS enhanced secretion both at intermediate (nano- to micromolar) and vanishingly low (<10 pM) Ca²⁺ concentrations. At optimal Ca²⁺ (10 M) the effect of GTPS was greatly reduced. We investigated whether the secretory response to GTP analogues was mediated by any of three enzyme systems regulated by GTP-binding proteins, i.e. generation of cyclic AMP by adenylate cyclase, of diacylglycerol by phospholipase C and of arachidonic acid by phospholipase A₂. The involvement of these messenger systems could be excluded as (i) cyclic AMP only had minor, Ca²⁺ dependent effects, (ii) phospholipase C was not activated in the absence of Ca²⁺ and insulin secretion due to the phorbol ester TPA displayed a different Ca²⁺ dependency, (iii) arachidonic acid did not elicit Ca²⁺ independent insulin secretion. These results, taken together with the finding that insulin secretion due to Ca²⁺ or TPA is attenuated by the inhibitory guanine nucleotide GDPS, suggest the existence of a regulatory site in exocytosis which is sensitive to guanine nucleotides.Abbreviations InsP₃ inositol trisphosphate - Ptd-InsP₂ phosphatidylinositol 4,5-bisphosphate - GTPS guanosine 5-(3-O-thio)triphosphate - GDPS guanosine 5-(2-O-thio)diphosphate - Gpp(NH)p guanyl-5-yl imidodiphosphate - TPA 12-O-tetradecanoylphorbol-13-acetate - OAG 1-oleoyl-2-acetylglycerol - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - EGTA (ethylenebis(oxyethylenenitrilo)tetraacetic acid - DAG diacylglycerol - [Ca²⁺]_i cytosolic free Ca²⁺ concentration     

A soluble calcium-binding protein from the terrestrial annelidLumbricus terrestris L.

Summary Soluble calcium-binding proteins (SCBP) considerably different from calmodulin were purified from the body wall muscle of the earthwormLumbricus terrestris. Three isoforms were obtained with similar UV absorption spectra and amino acid compositions and an apparent molecular weight close to 20 kDa. They can be distinguished by their histidine and proline content and by their peptide maps. The tissue content, as determined by quantitative ELISA varies individually from 0.1 to 0.3 mmol kg^–1. The calcium-binding property can be demonstrated by Ca²⁺-dependent electrophoretic mobility shift and⁴⁵Ca²⁺ autoradiography on nitrocellulose sheets. The apparentK _D values for the SCBP-Ca²⁺ complex is approximately 10^–7 mol l^–1 as revealed by euquilibrium and flow dialysis experiments. In the presence of 1 mmol l^–1 MgCl₂ the maximum binding capacity of SCBP was determined to be either 2 mol Ca²⁺ mol^–1 protein (SCBP₂) or 3 mol Ca²⁺ mol^–1 protein (SCBP₃). Preliminary studies concerning the functional role of SCBP indicate that it facilitates the diffusion of Ca²⁺ ions by a factor of 2 and is capable of inhibiting the ATPase of isolated body wall muscle actomyosin. The results reveal that earthworm SCBP are similar to vertebrate parvalbumin and to SCBP characterized from aquatic invertebrates.Abbreviations ABTS 2,2-azino-di-(3-ethyl)-benzothiazolinsulfonate - CN-PDE 3:5-cyclic nucleotide-phosphodiesterase - DEAE diethylaminoethyl - EGTA ethyleneglycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - ELISA enzyme linked immuno sorbent assay - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - HPLC high performance liquid chromatography - HRP horseradish peroxydase - PAGE polyacrylamide gel electrophoresis - PBS phosphate buffered saline - P _i inorganic phosphate - PMSF phenylmethylsulfonyl fluoride - SCBP soluble calcium-binding protein - SDS sodium dodecyl sulphate - SPDP N-succininydyl-3-(2-pyridyldithio)propionate - SR sarcoplasmic reticulum - Tris tris(hydroxymethyl)-aminomethane - UV ultraviolet     

Investigation of the Ca2+-dependent interaction of trifluoperazine with S100a: A19F NMR and circular dichroism study

S100a is a heterodimeric, acidic calcium-binding protein that interacts with calmodulin antagonists in a Ca²⁺-dependent manner. In order to study the behavior of the hydrophobic domain on S100a when bound to Ca²⁺, its interaction with trifluoperazine (TFP) was investigated using¹⁶F nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy. The dissociation constant (K _d) values of TFP, as estimated from the chemical shifts of¹⁹F NMR, were 191 and 29 m in the absence and presence of Ca²⁺, respectively, and were similar to those previously reported for S100b. However, the TFP linewidth in the presence of Ca²⁺-bound S100a was 65 Hz greater than in the presence of Ca²⁺-bound S100b. This suggests a slower TFP exchange rate for S100a than for S100b. Thus, the TFP linewidths observed for each isoform may reflect differences in structural and modulatory properties of the Ca²⁺-dependent hydrophobic domains on S100a and S100b. Additionally, the presence of magnesium had no effect on the observed Ca²⁺-induced TFP spectral changes in S100a solutions. Circular dichroism studies indicate that Ca²⁺ induces a small transition from -helix to random coil in S100a; in contrast, the opposite transition is reported for calmodulin (Hennesseyet al., 1987). However, TFP did not significantly alter the secondary structure of Ca²⁺-bound S100a; this observation is similar to the effect of TFP on Ca²⁺-bound calmodulin and troponin C (Shimizu and Hatano, 1984; Gariépy and Hodges, 1983). It is, therefore, proposed that TFP binds to a hydrophobic domain on S100a in a fashion similar to other calcium-modulated proteins.     

Evolutionary temperature adaptation of fish sarcoplasmic reticulum

Summary Sarcoplasmic reticulum has been isolated from the white muscle of 15 species of teleost fish adapted to diverse thermal environments. Evidence has been obtained that the Ca²⁺-dependent ATPase of fish sarcoplasmic reticulum has undergone evolutionary modification for function at different temperatures. Compared with tropical fish, cold adapted species have higher rates of Ca²⁺ transport and Ca²⁺-ATPase activities at low temperatures. Most species have linear Arrhenius plots over the temperature range 0–30°C. Activation enthalpies (H ) of the ATPase ranged from 53–190 kJ mol^–1 and were positively correlated with environment temperature. Activation entropy (S ) varied from negative values in cold adapted species to positive values in tropical fish.In contrast to the Ca²⁺-ATPase, the basal ATPase of fish sarcoplasmic reticulum showed no relationship between either ATPase activity or thermodynamic activation parameters and environmental temperature.Only the Ca²⁺-dependent ATPase is coupled to Ca²⁺ transport. The percentage of total ATPase activity which is Ca²⁺ activated is higher at low temperatures in cold than in warm adapted species. For example, ratios of Ca²⁺-dependent/total ATPase at 2°C varied from 80–98% in Arctic, Antarctic and North Sea species to only 2–50% in various tropical fish. Above 20°C, similar ratios in the range 80–98% were obtained for all species. The nature of the basal ATPase and mechanisms of temperature adaptation of fish sarcoplasmic reticulum are discussed.Abbreviations ET environmental temperature - EGTA ethylene glycol-bis (-aminolethyl ether)-N, N-tetraacetic acid - HEPES N-2-hydroxylpiperazine-N-2-ethanesulfonic acid - SR sarcoplasmic reticulum     

Ca2+-Independent Autophosphorylation of Postsynaptic Density-Associated Ca2+/Calmodulin-Dependent Protein Kinase

A major protein in the postsynaptic density fraction is -CAM kinase II, the -subunit of the Ca²⁺/calmodulin-dependent protein kinase. Autophosphorylation of the postsynaptic density-associated CaM kinase II is likely to be a crucial event in the induction of activity-dependent synaptic modification. This study focuses on the regulation and consequences of Ca²⁺-independent autophosphorylation of the enzyme. In isolated postsynaptic densities, a sub-stochiometric level of autophosphorylation in the presence of Ca²⁺ is sufficient to trigger maximal Ca²⁺-independent autophosphorylation of -CaM Kinase II. A major fraction of the sites phosphorylated in the absence of Ca²⁺ can be dephosphorylated by the endogenous phosphatase activity in the preparation. Ca²⁺-independent autophosphorylation is correlated with a drastic decrease in calmodulin binding to postsynaptic densities. This may represent a physiological mechanism that lowers the calmodulin trapping capacity of the organelle, thus increasing the availability of calmodulin to other elements within a spine.