Mixture Of Ca2 Research Articles

Engineering the Performance of Artificial Inclusion Bodies Built of Catalytic β-Galactosidase

One of the most critical bottlenecks in the application of industrial enzymes is the preservation of protein stability throughout the catalytic reaction, which often requires protein engineering and/or process optimization. In this context, we have designed and deeply characterized an efficient, stable, and reusable enzymatic platform based on the Escherichia coli β-galactosidase. The enzyme was assembled in vitro, by using divalent cations as molecular linkers, as stable protein microparticles showing catalytic activity. In this assembled microstructure, β-galactosidase exhibits a particular conformation within the microparticles, sharing structural traits (a high cross-parallel beta-sheet content) with the bacterial inclusion bodies and secretory amyloids from the mammalian endocrine system. This fact confers enhanced thermal stability compared to the soluble protein version and ensures high reusability in industry-oriented processes. On the other hand, among the catalog of cations tested as molecular linkers, a mixture of Ca2+ and Mg2+ offers the best performance to the catalytic particle. Altogether, these data offer clues for the application of a self-immobilized enzymatic platform with transversal applicability and enormous potential in biotechnology and biomedicine.

Condensation of iso-humulone in solution and at hydrophobic surfaces

Iso-humulone is an essential component for beer derived from hop, which is able to improve foam stability, increase bitterness and prolong shelf life, but its physiochemical properties have not been fully characterised. There is a limited amount of knowledge on how iso-humulone behaves in solution and at interfaces. In this work, the properties of iso-humulone are characterised in solution and at hydrophobic surfaces. The results show that iso-humulone is able to self-associate both in bulk solution and at the interface. The presence of ions typically found in beer (the mixture of Ca2+, K+, Mg2+ and Na+) increases the aggregation and adsorbed mass at the interface. Cryo-TEM confirms the presence of a liquid-like phase with nano-droplets with diameters of 20−90 nm. The results suggest that the droplets are spontaneously formed, have a limited growth through Ostwald ripening and can be dissolved upon dilution.

Localization of sperm intracellular Ca2+ keeps fertilizability in the newt vas deferens.

Sperm intracellular Ca2+ is crucial for the induction of sperm-egg interaction, but little is known about the significance of Ca2+ maintenance prior to induction. In sperm of the newt Cynops pyrrhogaster, intracellular Ca2+ is localized to the midpiece during storage in the vas deferens, while extracellular Ca2+ is influxed in modified Steinberg's salt solution to promote a spontaneous acrosome reaction related to the decline of sperm quality. In the present study, sperm from the vas deferens were loaded with the Ca2+ indicator Fluo8H, and changes in Ca2+ localization in modified Steinberg's salt solution were examined. Calcium ions expanded from the cytoplasmic area of the midpiece to the entire tail in most sperm during a 1-h incubation and localized to the principal piece in some sperm within 24 h. Similar changes in Ca2+ localization were observed in reconstructed vas deferens solution that included ions and pH at equivalent levels to those in the vas deferens fluid. Sperm with Ca2+ localization in the entire tail or the principal piece weakened or lost responsiveness to sperm motility-initiating substances, which trigger sperm motility for fertilization, but responded to a trigger for acrosome reaction. The change in Ca2+ localization was delayed and transiently reversed by ethylene glycol tetraacetic acid or a mixture of Ca2+ channel blockers including Ni2+ and diltiazem. These results suggest that C. pyrrhogaster sperm localize intracellular Ca2+ to the midpiece through Ca2+ transport in the vas deferens to allow for responses to sperm motility-initiating substances.

Magnesium substitution in calcium and strontium fluoro-phospho-aluminosilicate glasses by multinuclear 19F, 31P, 27Al, and 29Si MAS-NMR spectroscopy

Abstract Two series of glasses were produced by systematically substituting magnesium for calcium and strontium in the glass compositions, 4.5SiO2. 3Al2O3. 1.5P2O5. (3-x)CaO/SrO. xMgO. (2-y)CaF2/SrF2. yMgF2, 0 ≥ x ≥ 3, 0 ≥ y ≥ 2 and these glasses were characterised by 19F, 31P, 27Al, and 29Si MAS-NMR spectroscopy. The substitution of magnesium for calcium and strontium resulted in the gradual formation of F-Mg(n) species over the F-Ca(n) and F-Sr(n) with additional loss of Al F species. The 31P and 27Al MAS-NMR spectra showed the presence of Al-O-PO33− species charge balanced by a mixture of Ca2+ and Mg2+ or Sr2+ and Mg2+. In addition to that, the 31P MAS-NMR spectrum of the fully magnesium substituted glass showed the presence of amorphous magnesium pyrophosphate, Mg2P2O7. Al(IV) was the major Al species with small amount of Al(V) and Al(VI), but the amount of Al(V) increased with increase in magnesium substitution. Moreover, the fully magnesium substituted fluoro-phospho-aluminosilicate glass resulted in the more polymerised silicate network with increased number of the bridging oxygen on SiO4 tetrahedra.

Porous Methyltrimethoxysilane Coated Nanoscale‐Hydroxyapatite for Removing Lead Ions from Aqueous Solutions

The aim of this study was to synthetize new porous nanoparticles based on methyltrimethoxysilane coated hydroxyapatite (MTHAp) for lead removal form aqueous solutions. The morphological and compositional analysis of MTHAp were investigated by X‐ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM) equipped with an energy dispersive X‐ray spectrometer (EDS). Removal experiments of Pb2+ ions were carried out in aqueous solutions with controlled concentration of Pb2+ at a fixed pH value of 3 and 5 respectively. After the removal experiment of Pb2+ ions from solutions, porous hydroxyapatite nanoparticles were transformed into PbMTHAp_3 and PbMTHAp_5 via the adsorption of Pb2+ ions followed by a cation exchange reaction. The X‐ray diffraction spectra of PbMTHAp_3 and PbMTHAp_5 revealed that the powders, after removal of the Pb2+ ions, were a mixture of Ca2.5Pb7.5(PO4)6(OH)2, Pb2Ca4(PO4)2(SiO4), and Ca10(PO4)6(OH)2. Our results demonstrate that the porous hydroxyapatite nanoparticles can be used as an adsorbent for removing Pb2+ ions from aqueous solutions.

Towards the establishment of a general rate law for gypsum nucleation

Gypsum nucleation kinetics from a wide range of chemical compositions (1.45<Ca2+/SO42-<115), ionic strengths (I=2.5–10m) and saturation state with respect to gypsum (Ωgyp=1.07–8.4) were examined in batch experiments containing mixtures of Ca2+-rich Dead Sea brine and SO42--rich seawater with or without addition of extra Na2SO4 and CaCl2·2H2O. The induction times attained in the present study were compiled together with literature values from experiments carried out under significantly different conditions (synthetic NaCl solutions; I=0.09–6.3 m; Ca2+/SO42-=1; Ωgyp:1.59–7.76). Despite the variability in the experimental solutions, a single rate law based on classic nucleation theory was formulated to describe the induction times from more than 80 experiments:logTind=log13.17·Cs·exp-7.08ln2Ω+0.072·Cs·exp-1.426ln2Ωwhere Tind is the induction time, Cs is the solubility of gypsum and Ω is the saturation state with respect to gypsum.The rate law provides Tind for gypsum precipitation from aqueous solutions at 25°C, containing no synthetic antiscalants or catalysts, within a 95% confidence interval within a factor of 5.Based on this rate law, we show that at present most of the precipitation of gypsum from the Dead Sea brine occurs following significant evaporation in the industrial evaporation ponds and not in the Dead Sea itself. Whereas Tind in Dead Sea brines is very long (on the order of 3years), the evaporation of brine in the industrial ponds leads to increased Ω values, and thus to short Tind in the order of a few days. However, if seawater or reject brine from seawater desalinization will be introduced to the Dead Sea to restore its declining level, Tind will be significantly reduced and gypsum nucleation and precipitation will occur. For evaporated seawater, the proposed rate law predicts that even though saturation is obtained when seawater is evaporated by a factor of 2.8, gypsum will nucleate at reasonable times (few years) only when seawater are evaporated by a factor of ∼3.3.

Norepinephrine Causes a Biphasic Change in Mammalian Pinealocye Membrane Potential: Role of α1B-Adrenoreceptors, Phospholipase C, and Ca2+

Perforated patch clamp recording was used to study the control of membrane potential (V(m)) and spontaneous electrical activity in the rat pinealocyte by norepinephrine. Norepinephrine did not alter spiking frequency. However, it was found to act through α(1B)-adrenoreceptors in a concentration-dependent manner (0.1-10 μM) to produce a biphasic change in V(m). The initial response was a hyperpolarization (∼13 mV from a resting potential of -46 mV) due to a transient (∼5 sec) outward K(+) current (∼50 pA). This current appears to be triggered by Ca(2+) released from intracellular stores, based on the observation that it was also seen in cells bathed in Ca(2+)-deficient medium. In addition, pharmacological studies indicate that this current was dependent on phospholipase C (PLC) activation and was in part mediated by bicuculline methiodide and apamin-sensitive Ca(2+)-controlled K(+) channels. The initial transient hyperpolarization was followed by a sustained depolarization (∼4 mV) due to an inward current (∼10 pA). This response was dependent on PLC-dependent activation of Na(+)/Ca(2+) influx but did not involve nifedipine-sensitive voltage-gated Ca(2+) channels. Together, these results indicate for the first time that activation of α(1B)-adrenoreceptors initiates a PLC-dependent biphasic change in pinealocyte V(m) characterized by an initial transient hyperpolarization mediated by a mixture of Ca(2+)-activated K(+) channels followed by a sustained depolarization mediated by a Ca(2+)-conducting nonselective cation channel. These observations indicate that both continuous elevation of intracellular Ca(2+) and sustained depolarization at approximately -40 mV are associated with and are likely to be required for activation of the pinealocyte.

A Direct Redox Regulation of Protein Kinase C Isoenzymes Mediates Oxidant-induced Neuritogenesis in PC12 Cells

In this study, we have used the PC12 cell model to elucidate the mechanisms by which sublethal doses of oxidants induce neuritogenesis. The xanthine/xanthine oxidase (X/XO) system was used for the steady state generation of superoxide, and CoCl(2) was used as a representative transition metal redox catalyst. Upon treatment of purified protein kinase C (PKC) with these oxidants, there was an increase in its cofactor-independent activation. Redox-active cobalt competed with the redoxinert zinc present in the zinc-thiolates of the PKC regulatory domain and induced the oxidation of these cysteine-rich regions. Both CoCl(2) and X/XO induced neurite outgrowth in PC12 cells, as determined by an overexpression of neuronal marker genes. Furthermore, these oxidants induced a translocation of PKC from cytosol to membrane and subsequent conversion of PKC to a cofactor-independent form. Isoenzyme-specific PKC inhibitors demonstrated that PKCepsilon plays a crucial role in neuritogenesis. Moreover, oxidant-induced neurite outgrowth was increased with a conditional overexpression of PKCepsilon and decreased with its knock-out by small interfering RNA. Parallel with PKC activation, an increase in phosphorylation of the growth-associated neuronal protein GAP-43 at Ser(41) was observed. Additionally, there was a sustained activation of extracellular signal-regulated kinases 1 and 2, which was correlated with activating phosphorylation (Ser(133)) of cAMP-responsive element-binding protein. All of these signaling events that are causally linked to neuritogenesis were blocked by antioxidant N-acetylcysteine (both L and D-forms) and by a variety of PKC-specific inhibitors. Taken together, these results strongly suggest that sublethal doses of oxidants induce neuritogenesis via a direct redox activation of PKCepsilon.

Multicomponent Titration of Calcium + Magnesium Mixtures Employing a Potentiometric Electronic‐Tongue

A fast novel potentiometric titration procedure is proposed, in which the detection system is formed by a potentiometric electronic tongue. The titration consists in a reduced number of fixed titrant additions to the sample and the recording of the potentials of an array of Ion Selective Electrodes. The obtained data matrix is entered to an Artificial Neural Network response model, previously trained to furnish concentrations of a multicomponent mixture. The principle is demonstrated with automated EDTA titration of mixtures of Ca2+ and Mg2+ at fixed pH 8.5. In this case, five 2 ml fixed volume additions plus the readings of four sensors were adequate for the resolution of the mixture. In the conditions used, titration was feasible up to 3.3 mM total ion concentration, the precision was estimated as 3.20% RSD for Ca2+ and 2.76% RSD for Mg2+ (n=5), and the detection limits 0.16 mM Ca2+ and 0.26 mM Mg2+. The procedure was applied to mineral waters and compared with reference methods (correlation=0.92 for Ca2+ and correlation=0.89 for Mg2+, n=14).

Both Ca2+‐permeable and ‐impermeable AMPA receptors contribute to primary synaptic drive onto rat dorsal horn neurons

Blockade of Ca2+-permeable AMPA receptors in the rat spinal cord diminishes the development of hyperalgesia and allodynia associated with peripheral injury. Cobalt uptake studies reveal that Ca2+-permeable AMPA receptors are expressed by some substance P receptor-expressing (NK1R+) neurons in lamina I, as well as other neurons throughout the superficial dorsal horn. Selective elimination of NK1R+ neurons in lamina I and lamina III/IV of the dorsal horn also suppresses development of hyperalgesia and allodynia. These observations raise the possibility that Ca2+-permeable AMPA receptors contribute to excitatory synaptic drive onto the NK1R+ neurons associated with allodynia and hyperalgesia. The first synapse in the pain pathway is the glutamatergic excitatory drive from the primary afferent fibres onto dorsal horn neurons. Therefore, we tested whether Ca2+-permeable AMPA receptors are located on lamina I and lamina III/IV NK1R+ neurons postsynaptic to primary afferent fibres, using inward rectification and polyamine toxins for receptor identification. We examined three different populations of dorsal horn neurons; lamina I NK1R+ neurons, including projection neurons, and non-NK1R+ (NK1R-) neurons including interneurons, and lamina III/IV NK1R+ neurons, believed to contribute to the low-threshold mechanosensory pathway. The majority of synapses in all three groups had rectification indices less than 1.0 and greater than 0.4, indicating that the AMPA receptors at these synapses are a mixture of Ca2+-permeable and -impermeable forms. Lamina III/IV NK1R+ neurons and lamina I NK1R- neurons have a significantly higher proportion of postsynaptic Ca2+-permeable AMPA receptors than lamina I NK1R+ neurons. Thus synaptically positioned Ca2+-permeable AMPA receptors directly contribute to low-threshold sensory afferent drive into the dorsal horn, and can mediate afferent input onto interneurons such as GABAergic neurons. These receptors also contribute to high-threshold primary afferent drive onto NK1R+ neurons in the superficial dorsal horn, but do so less consistently.

Effects of a gerF (lgt) mutation on the germination of spores of Bacillus subtilis.

One of the proteins of the membrane-bound receptors that recognize individual nutrients that trigger germination of spores of Bacillus subtilis contains the recognition sequence for diacylglycerol addition to a cysteine residue near the protein's N terminus. B. subtilis spores lacking the gerF (lgt) gene that codes for prelipoprotein diacylglycerol transferase exhibited significantly slowed germination in response to nutrient germinants as found previously, but germination of gerF spores with a mixture of Ca2+ and dipicolinic acid or with dodecylamine was normal, as was the spontaneous germination of gerF spores lacking all nutrient germinant receptors. The deleterious effects of the gerF mutation on nutrient germination were highest on germination triggered by the GerA nutrient receptor and were less so (but still significant) on germination triggered by the GerB nutrient receptor. However, there was little, if any, effect on GerK nutrient receptor-mediated spore germination. As predicted from the latter results, replacement by alanine of the cysteine residue to which diacylglycerol is thought to be added to these nutrient receptors had a large effect on GerA receptor function, less effect on GerB receptor function, and little, if any, effect on GerK receptor function.

Pore properties and ionic block of the rabbit epithelial calcium channel expressed in HEK 293 cells.

We have used the whole-cell patch-clamp technique to analyse the permeation properties and ionic block of the epithelial Ca2+ channel ECaC heterologously expressed in human embryonic kidney (HEK) 293 cells. Cells dialysed with 10 mM BAPTA and exposed to Ca2+-containing, monovalent cation-free solutions displayed large inwardly rectifying currents. Their reversal potential depended on the extracellular Ca2+ concentration, [Ca2+]o. The slope of the relationship between reversal potential and [Ca2+]o on a logarithmic scale was 21 +/- 4 mV, compared with 29 mV as predicted by the Nernst equation (n = 3-5 cells). Currents in mixtures of Ca2+ and Na+ or Ca2+ and Ba2+ showed anomalous mole fraction behaviour. We have described the current-concentration plot for Ca2+ and Na+ by a kinetic permeation model, i.e. the "step" model. Extracellular Mg2+ blocked both divalent and monovalent currents with an IC50 of 62 +/- 9 microM(n = 4) in Ca2+-free conditions and 328 +/- 50 microM (n = 4-9) in 100 microM Ca2+ solutions. Mono- and divalent currents through ECaCs were blocked by gadolinium, lanthanum and cadmium, with a blocking order of Cd2+ >> Gd3+ > La3+. We conclude that the permeation of monovalent and divalent cations through ECaCs shows similarities with L-type voltage-gated Ca2+ channels, the main differences being a higher Ca2+ affinity and a significantly higher current density in micromolar Ca2+ concentrations in the case of ECaCs.

L-, N- and T- but neither P- nor Q-type Ca2+ channels control vasopressin-induced Ca2+ influx in magnocellular vasopressin neurones isolated from the rat supraoptic nucleus.

1. The role of voltage-dependent Ca2+ channels during vasopressin and oxytocin actions on their respective neurones has been analysed by measuring intracellular Ca2+ concentration ([Ca2+]i) in individual, freshly dissociated magnocellular neurones from rat supraoptic nucleus (SO) using microspectrofluorimetry. 2. Pre-incubation of vasopressin-sensitive neurones with Cd2+ (100 microM), a non-discriminatory high-voltage-activated Ca2+ channel antagonist, or Ni2+ (50 microM), a blocker of T-type Ca2+ current, reduced [Ca2+]i responses by 77 and 19%, respectively. When Cd2+ was given together with Ni2+, the response was blocked by 92%. Similarly, when Ni2+ was pre-incubated with Cd2+, the response was blocked by approximately 84%. 3. Exposure of vasopressin sensitive neurones to a specific Ca2+ channel blocker, nicardipine (L-type) reduced vasopressin responses by 48% at 1 microM and 62% at 5 microM. Similarly, omega-conotoxin GVIA (omega-CgTX, N-type; 500 nM) inhibited the response by 46% with a stronger inhibition (75%) at 800 nM. By contrast, neither omega-agatoxin IVA (omega-Aga IVA; 300 nM), which blocks both P- and Q-type channels, nor synthetic omega-conotoxin MVIIC (omega-MVIIC; 100 or 500 nM), a Q-type blocker, affected vasopressin-induced [Ca2+]i responses. These antagonists, given together (nicardipine 5 microM + omega-CgTX 800 nM + omega-Aga IVA 300 nM), decreased vasopressin-induced [Ca2+]i responses by 76%. 4. In vasopressin-sensitive neurones, the presence of both nicardipine and omega-CgTX, reduced the K(+)-evoked [Ca2+]i increase by 61%. This blockade was increased by a further 21% with omega-Aga IVA, suggesting that N-, L- and P-type channels contribute to the depolarization-induced [Ca2+]i rise. In addition, omega-MVIIC alone reduced the K(+)-evoked [Ca2+]i release by 24%. Also the remaining K+ responses were further reduced by 60% when pre-incubated with L-N- and P-type blockers, suggesting the involvement of Q-type channels. 5. In oxytocin-sensitive neurones, the peak amplitude of the [Ca2+]i response was not affected by Cd2+ alone, by combined Cd2+ and Ni2+, or by the mixture of nicardipine, omega-CgTX and omega-Aga IVA. By contrast, the responses evoked by depolarization with K+ were blocked by Cd2+. Both nicardipine and omega-CgTX blocked 65% of K+ response and an additional block of approximately 18% was obtained with omega-Aga IVA, suggesting the involvement of L-, N- and P-type channels. In combination, these antagonists strongly inhibited (approximately 80% reduction) the K+ responses. Further reduction to 18% was made by the Q-type blocker omega-MVIIC. Pre-incubation with L-, N- and P-type blockers caused an additional block of 71%. 6. Some supraoptic neurones (5-10%) responded to both vasopressin and oxytocin, with only the [Ca2+]i responses induced by vasopressin blocked (> 90% inhibition) by the mixture of Ca2+ channel antagonists. 7. In conclusion, both vasopressin and oxytocin magnocellular SO neurones have been shown to express T-, L-, N-, P-, Q- and R-type Ca2+ channels in their somata. Our results show that the vasopressin-induced [Ca2+]i increase in vasopressin-sensitive neurones is mediated by L-, N- and T-type Ca2+ channels and not by P- and Q-type channels; Ca2+ channels are not involved in oxytocin action on oxytocin-sensitive neurones and L-, N-, P- and Q-type channels control the K(+)-induced [Ca2+]i increase in SO neurones.

Thermal Stability of Whey Protein Concentrate Mixtures: Aggregate Formation

Solutions (17% TS) of whey protein concentrate (65% protein) were dialyzed against simulated milk ultrafiltrate containing 0 to 9mM total Ca2+. The dialyzed solutions were heated at 66 or 71°C for 120min to study the effect of Ca2+ on the heat denaturation and aggregation of whey proteins. As Ca2+ decreased, the whey protein concentrate solutions formed more soluble aggregates and fewer insoluble precipitates; the amount of α-LA relative to the β-LG associated with the soluble aggregates also increased. Protein aggregates, as shown by electron microscopy of the Ca2+-adjusted solutions, became smaller and less densely packed as Ca2+ decreased. The effects of addition of low heat NDM or a mixture of Ca2+ and Na caseinate to the whey protein concentrate solution (17% TS) and heat treatment at 71°C for 120min on whey protein denaturation and aggregate formation were also investigated. Compared with the whey protein concentrate solution (17% TS), whey protein denaturation was much lower when low heat NDM was added to the solution, but not when a mixture of Ca2+ and Na caseinate was added. Electron micrographs showed that the whey protein aggregates that formed upon heating the mixture of whey protein concentrate and low heat NDM at 71°C were smaller and less dense, and the micellar appendages were more compact, than those in the whey protein (17% TS) or low heat NDM (27% TS) solutions. The micrographs of the mixture of whey protein concentrate and caseinate were not comparable with either that of low heat NDM or of the mixture of whey protein concentrate and NDM.

Calcium-release-activated calcium influx in endothelium.

Signaling pathways activated by the tachykinin substance P (SP) were investigated in pig coronary artery endothelial cells (PCAECs). Single cells were obtained after enzymatic digestion of coronary arteries. Intracellular Ca2+ ([Ca2+]i) was measured from fura-2 fluorescence while membrane potential or ionic current was measured using patch-clamp techniques. In physiological saline solution, SP induced hyperpolarizations or outward currents which coincided with biphasic [Ca2+]i increases representing store release of Ca2+ and Ca2+ influx. Single channel recording protocols showed that both sources of Ca2+ activated a small conductance K+ channel, resulting in cell hyperpolarization. When outward currents were blocked by d-tubocurare, Cs+, or BAPTA, an inward current was unmasked. Ion substitution protocols showed that the SP-induced inward current was (1) carried by a mixture of Ca2+ and Na+, (2) blocked by La3+, and (3) inactivated by high extracellular [Ca2+]. Tyrosine kinase inhibitors also blocked the inward current. The same current was activated by bath application of BHQ, an inhibitor of the endoplasmic reticulum Ca2+ ATPase, or by cell dialysis with IP3. These results suggest that the plateau phase of the agonist-activated [Ca2+]i increase in PCAECs reflects Ca2+ entry through a depletion-activated Ca2+ channel. The characteristics of this channel are compared to those of Ca2+ channels found in other nonexcitable cells.

Physiological properties of skinned fibres from normal and dystrophic (Duchenne) human muscle activated by Ca2+ and Sr2+.

1. Contractile activation properties of various types of normal and dystrophic (Duchenne muscular dystrophy, DMD) human muscle fibres were investigated using mechanically skinned fibres activated in Ca2(+)- and Sr2(+)-buffered solutions at room temperature (21-25 degrees C). 2. The majority of the normal human muscle fibres (18/22; 82%) could be classified according to the Ca2(+)- and Sr2(+)-activation characteristics in the same three major groups as other mammalian skeletal muscle fibres studied previously: slow-twitch (8/22; 36%), fast-twitch (3/22; 14%) and fast-twitch intermediate (7/22; 32%), which correspond to the three major histological fibre types I, II B and IIA respectively. 3. Of these three major groups only the slow-twitch (type I; 14/24; 58%) and the fast-twitch intermediate (type IIA; 3/24; 13%) fibres were found in the DMD muscle, indicating that fast-twitch fibres of type IIB were in very low proportion in DMD muscle. 4. The DMD muscle contained a new group of fibres (4/24; 17%) with different Ca2(+)- and Sr2(+)-activation characteristics from the three major histological types. This group of fibres is likely to be of embryonic type. 5. The maximum tension development ability of DMD fibres was less than 20% of that in normal fibres with the exception of some slow-twitch fibres which could produce near normal tension. 6. A significant proportion of normal (4/22; 18%) and DMD (3/24; 13%) fibres were found to have a mixture of Ca2(+)- and Sr2(+)-activation characteristics. This can be explained by co-existence of various myofibrillar protein isoforms in different proportions with regulatory functions in the same individual fibre. 7. The results demonstrate that DMD leads to marked diminution in the ability of most individual skeletal muscle fibres to develop tension, and causes changes in the overall fibre-type distribution in afflicted muscles.

Demonstration of two protein kinases in extracts of Legionella micdadei.

Protein kinases I (PK I) and II (PK II) were purified 253- and 13.5-fold, respectively, from an extract of sonically disrupted cells of Legionella micdadei by ion-exchange chromatography on QAE-Sephadex, by histone affinity chromatography, and by HPLC-gel filtration chromatography. Both enzymes catalysed the phosphorylation of calf thymus histones, with a Km of 2.7 mg ml-1 for PK I and 2.9 mg ml-1 for PK II. Histone H2b was the best protein kinase substrate for both PK I and PK II. The pH optima were 6.8 and 7.0 for PK I and PK II respectively. The Km for ATP was 0.29 mM for PK I and 0.33 mM for PK II. PK II activity was stimulated by either cAMP or cGMP, whereas PK I was inhibited by both cyclic nucleotides. The activity of PK I was unaffected by addition of calmodulin, diacylglycerol and mixtures of Ca2+ and acidic phospholipids, but these additions increased PK II activity threefold. The activity of PK II was stimulated by spermine and spermidine, but PK I was inhibited by these compounds. PK I and PK II were both strongly inhibited by heparin.

Permeation and interaction of divalent cations in calcium channels of snail neurons.

We have studied the current-carrying ability and blocking action of various divalent cations in the Ca channel of Lymnaea stagnalis neurons. Changing the concentration or species of the permeant divalent cation shifts the voltage dependence of activation of the Ca channel current in a manner that is consistent with the action of the divalent cation on an external surface potential. Increasing the concentration of the permeant cation from 1 to 30 mM produces a twofold increase in the maximum Ca current and a fourfold increase in the maximum Ba current; the maximum Ba current is twice the size of the maximum Ca current for 10 mM bulk concentration. Correcting for the changing surface potential seen by the gating mechanism, the current-concentration relation is almost linear for Ba2+, and shows only moderate saturation for Ca2+; also, Ca2+, Ba2+, and Sr2+ are found to pass through the channel almost equally well. These conclusions are obtained for either of two assumptions: that the mouth of the channel sees (a) all or (b) none of the surface potential seen by the gating mechanism. Cd2+ blocks Lymnaea and Helix Ca channels at concentrations 200 times smaller than those required for Co2+ or Ni2+. Ca2+ competes with Cd2+ for the blocking site; Ba2+ binds less strongly than Ca2+ to this site. Mixtures of Ca2+ and Ba2+ produce an anomalous mole fraction effect on the Ca channel current. After correction for the changing surface potential (using either assumption), the anomalous mole fraction effect is even more prominent, which suggests that Ba2+ blocks Ca current more than Ca2+ blocks Ba current.