Aggregation of sponge cells: 22. Species-specific reactivity of a lectin from the sponge Geodia cydonium

Single cells of the marine sponge Geodia cydonium aggregate species-specifically in the presence of a soluble aggregation factor to form large cell clumps. A lectin isolated from the same sponge species does not cause agglutination of Geodia cells but agglutinates only cells from heterologous species (e.g. Tethya lyncurium, Hemimycale columella, Pellina semitubulosa, Cacospongia scalaris, Verongia aerophoba). The process of agglutination is independent of divalent cations (they do not affect the agglutination process at concentrations up to 50 mM), occurs at 2°C, causes a reduction in the viability of the cells and results in an inhibition of programmed syntheses. The observed differences between the properties of cell agglutination (effect of a lectin in a heterologous system) and cell aggregation (effect of an aggregation factor in the homologous system) is discussed. Cell aggregation is dependent upon the presence of an aggregation factor, the presence of cations and an incubation temperature 2̃0°C; cell aggregation results in a stimulation of programmed syntheses. Cell agglutination requires a heterologous macromolecule (e.g. lectin), it is independent of divalent cations and causes inhibition of programmed syntheses in the cells.     

Inactivation of endotoxin by Limulus amoebocyte lysate.

The inactivation of bacterial endotoxin by aqueous extracts (Limulus amoebocyte lysate) of the circulating blood cells (amoebocytes) of the horseshoe crab, Limulus polyphemus, is described. Active extracts were obtained by heating Limulus amoebocyte lystate (LAL) to 60°C for 20 min to denature the clotting enzyme, rendering the LAL incapable of gel formation in the presence of endotoxin. Endotoxin inactivation was assayed using the Limulus amoebocyte lysate test and by rabbit bioassay. Inactivation of endotoxin with heated extracts of LAL was suggestive of enzymatic mediation, as indicated by dependence on time, temperature, pH, and the kinetics of inactivation. Endotoxin inactivation occurred over a broad pH range, 4.5–8.5, with the optimum at a pH of 6.1. Temperature optima were between 37° and 50°C, with observed activity between 0° and 65°C. Ionized calcium was inhibitory to endotoxin inactivation with heated extracts of LAL, with partial inhibition at 0.001 m calcium and complete inhibition at 0.02 m calcium. Other divalent cations (Mg, Ba, Mn, and Cu) were also found to inhibit the inactivation of endotoxin. Similarities between the endotoxin-inactivating system of L. polyphemus and those described to be present in mammalian and lower vertebrate sera are discussed.     

5′-Nucleotidase from bovine brain cortex: effect of solubilization on enzyme kinetics and modulation

The kinetic characteristics and the EDTA inhibition of microsomal 5′-nucleotidase from bovine brain cortex were studied and compared with the properties of the enzyme solubilized with Lubrol WX. The K_m value after enzyme solubilization was not significantly different from that of the membrane-bound enzyme. Likewise, di- and trinucleotides performed a similar competitive inhibition of the two forms of the enzyme. In contrast, divalent cations inhibited the intact microsomal enzyme activity at the same concentrations in which they increased the soluble-enzyme activity. The solubilization of microsomal 5′-nucleotidase did not change the progressive and irreversible character of the EDTA inhibition, but the mechanism of the irreversible inhibition was different. The addition of divalent metal cations did not affect the irreversibility of either inhibition, even though the effect on the residual activities was different. The Arrhenius plot of the 5′-nucleotidase activity in intact microsomal fraction exhibited a well-defined break at 31 ± 0.1°C, whereas that of the solubilized enzyme was a straight line. It is concluded then that microsomal 5′-nucleotidase from bovine brain cortex does not require the membrane environment to express its activity, although the influence of this lipidic environment was evident in the differences observed in the enzyme activity modulation by EDTA, cations and temperature.     

Factors affecting the measurement of chemiluminescence in stimulated human polymorphonuclear leucocytes

Optimum conditions were established for the generation and measurement of luminoldependent chemiluminescence (CL) in human polymorphonuclear leucocytes (PMNL) stimulated with a variety of particulate and soluble agents. Several factors had a particular influence on the kinetics of CL stimulated by the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP). Two peaks, both azide-sensitive, were observed at 21°C and 25°C. but these increased in magnitude and merged t o give a single, early peak when the temperature was increased t o 37°C. Pre-exposure of PMNL to a buffer containing calcium was essential for the expression of both phases of fMLP-stimulated CL, while the second peak decreased dramatically if the cells were stored at 4°C for 4 hours before assay. In contrast, storage of PMNL at 4°C for up t o 8 hours in a buffer without divalent cations did not alter the kinetics or magnitude of CL induced by other stimuli, and had the benefit of minimizing the rate of cell aggregation. This study confirms that measurement of luminol-dependent CL in stimulated PMNL is a useful analytical tool, but shows that careful attention t o experimental design is required t o ensure that the observed CL provides a true measure of the parameter under investigation.     

米氏凯伦藻溶血毒素的溶血反应特征

探讨了温度、pH值、二价阳离子等对米氏凯伦藻(Karenia mikimotoi Hasen)溶血毒素溶血活性的影响,分析了米氏凯伦藻溶血毒素的溶血反应特征.结果表明,实验室培养米氏凯伦藻的溶血活性约为64.69±6.43 HU L-1,单个藻细胞的溶血活性为6.17±0.61×10-6 HU;在实验温度(0～37℃)下,溶血活性随温度的增加而增加;pH6.0时的溶血活性最高;Cu2+、Mg2+、Mn2+、Ca2+、Co2+、Zn2+和Hg2+等对米氏凯伦藻的溶血活性的影响不同.离子浓度为5 mmol/L时,Hg2+的抑制作用最强.高浓度Hg2+对红细胞的集合效应不但阻止了Hg2+进入血细胞诱导的溶血作用,而且阻止了毒素对细胞膜的破坏,但这种抑制作用可被EDTA消除.     

Calcium and magnesium effect on divalent cation deficientHydrilla verticillata thylakoid electron transport activity

The role of divalent cations like magnesium (Mg²⁺) and calcium (Ca²⁺) was irrvestigated on energy distribution process ofHydrilla verticillata thylakoids. Effect of these cations was tested on relative quantum yield of photosystem (PS) II catalyzed electron transport activity, room and liquid nitrogen temperature fluorescence emission properties and thylakoid light scattering characteristics. The electron transport activity was found to be stimulated in the presence of these cations in a light intensity independent manner. The concentration of cation required for maximum stimulation was nearly 10–12 mM. Comparatively, Ca²⁺ was more effective than Mg²⁺. Cation induced stimulation in electron transport activity was not accompanied by increase in chlorophylla fluorescence intensity either at room (25°C) or liquid nitrogen (77°K) temperatures. Furthermore, 540 nm absorption and 90° light scattering properties of thylakoids remained insensitive towards divalent cations. These facts together suggest that divalent cations inHydrilla thylakoids are not effective in supporting the excitation distribution between the interacting photosystem complexes.     

The effect of monovalent cations on calcium efflux in yeasts

The properties of the calcium efflux system in the yeast Saccharomyces cerevisiae were investigated. After growing the cells overnight in medium containing ⁴⁵Ca, the cells were transferred to medium containing glucose, Hepes buffer (pH 5.2) and monovalent cations. The presence of potassium or sodium in the medium induced efflux of calcium from the cells. The magnitude of the efflux was dependent on the concentration of these cations in the medium. The time course of calcium efflux was analyzed, and two types of exchangeable calcium pools, which turned over at different rates, were detected: ‘Fast turnover’ and ‘slow turnover’. Increase in the concentration of monovalent cations in the medium caused an increase in the fraction of cellular calcium which turned over at a fast rate, and activation of calcium efflux from the ‘slow turnover’ calcium pool. The specific changes in the parameters of calcium efflux induced by monovalent cations were different from those reported previously to be induced by divalent cations. Both processes, i.e. activation of calcium efflux by monovalent and by divalent cations, were found to be additive, indicating that they operate via different mechanisms. Experiments using the respiratory inhibitor Antimycin A, showed that stimulation of calcium efflux by monovalent cations is energy dependent. Lanthanum ions which are known to inhibit calcium influx into yeast cells, inhibitted the activation of calcium efflux by both divalent and monovalent cations. Determination of the cationic composition of the cells indicated that the stimulation of calcium efflux was accompanied by influx of potassium or sodium into the cells.     

The Role of Hydrated Divalent Metal Ions in the Bridging of Two Anionic Groups. An ab initio Quantum Chemical and Molecular Mechanics Study of Dimethyl Phosphate and Formate Bridged by Calcium and Magnesium Ions

Abstract

Ab initio quantum chemical (Gaussian82) and molecular mechanics (AMBER2.0) computational techniques are employed to investigate the interaction of twoanions (formate and dimethyl phosphate) and a central divalent metal cation (magnesium or calcium). These systems are models for the essential GDP binding unit of the G-proteins (e.g., EF-Tu or the ras oncogene proteins) and for protein/phospholipid interactions, both of which are mediated by divalent metal cations. Various levels of hydration are utilized to examine coordination of differences between magnesium and calcium ions. Two different orientations of formate and dimethyl phosphate in direct ion contact with a magnesium ion and two waters of hydration were energy minimized with both quantum and molecular mechanics techniques. The structures and energy differences between the two orientations determined by either of the computational techniques are similar. Magnesium ion has a strong propensity to assume six coordination whereas calcium ion preferentially assumes a coordination greater than six. Likewise, water molecules attached to magnesium ion are held more rigidly than those to calcium ion, thus calcium ion is more accommodating in the exchange of water for negative ligands.     

Energy transfer and the distribution of excitation energy in the photosynthetic apparatus of spinach chloroplasts

Equations are derived from our model of the photochemical apparatus of photosynthesis to show that the yield of energy transfer from Photosystem II to Photosystem I, ?_T(II→Iz), can be obtained from measurements on an individual sample of chloroplasts frozen to ?196 °C by comparing the sum of two specifically defined fluorescence excitation spectra with the absorption spectrum of the sample. Then, given that value of ?_T(II→I), the fraction of the quanta absorbed by the photochemical apparatus which is distributed initially to Photosystem I, α, can be determined as a function of the wavelength of excitation from the same fluorescence excitation spectra. The results obtained in this study of individual samples of chloroplasts frozen to ?196 °C in the absence of divalent cations, namely, that ?_T(II→I) varies from a minimum value of 0.10 when the Photosystem II reaction centers are all open to a maximum value of 0.25 when the centers are all closed and that α has a value of about 0.30 which is almost independent of wavelength for wavelengths shorter than 675 nm (α increases rapidly toward unity at wavelengths longer than 675 nm), agrees quite well with results obtained previously from comparative measurements of chloroplasts frozen to ?196 °C in the presence and absence of divalent cations.     

Kinetics of Ca2+ and Sr2+ uptake by yeast. Effects of pH,cations and phosphate

The uptake of Ca²⁺ and Sr²⁺ by the yeast Saccharomyces cerevisiae is energy dependent, and shows a deviation from simple Michaelis-Menten kinetics. A model is discussed that takes into account the effect of the surface potential and the membrane potential on uptake kinetics.The rate of Ca²⁺ and Sr²⁺ uptake is influenced by the cell pH and by the medium pH. The inhibition of uptake at low concentrations of Ca²⁺ and Sr²⁺ at low pH may be explained by a decrease of the surface potential.The inhibition of Ca²⁺ and Sr²⁺ uptake by monovalent cations is independent of the divalent cation concentration. The inhibition shows saturation kinetics, and the concentration of monovalent cation at which half-maximal inhibition is observed, is equal to the affinity constant of this ion for the monovalent cation transport system. The inhibition of divalent cation uptake by monovalent cations appears to be related to depolarization of the cell membrane.Phosphate exerts a dual effect on uptake of divalent cations: and initial inhibition and a secondary stimulation. The inhibition shows saturation kinetics, and the inhibition constant is equal to the affinity constant of phosphate for its transport mechanism. The secondary stimulation can only partly be explained by a decrease of the cell pH, suggesting interaction of intracellular phosphate, or a phosphorylated compound, with the translocation mechanism.     

Gated ion fluxes involved in photophobic responses of the blue-green alga,Phormidium uncinatum

The sensory transduction chain of photophobic responses in the blue-green alga, Phormidium uncinatum seems to involve a gating cation transport through membrane bound ion channels which provides an effective amplification.The calcium conducting ionophore A23187 inhibits the photophobic response totally and induces frequent reversals which resemble phobic responses but occur without any light stimulation. This indicates that the electrogenic ion conductance may depend on a gradient of divalent cations, esp. calcium. The calcium conductance during a photophobic response is further confirmed by the inhibitory effect of ruthenium red and lanthanum, blockers of the electrogenic calcium transport. In the case of lanthanum this inhibition is found at a concentration at which neither the number of motile filaments nor the average speed of movement is impaired.Incorporation of ionophores for monovalent cations (gramicidin and valinomycin) only partially impairs the response. Similarly, inhibition of the Na⁺/K⁺ pump by ouabain is less effective. Thus, the existence of a countercurrent of monovalent cations during the response, which has been described for e.g. ciliates, is yet obscure in blue-green algae.     

Neurotoxic unc-8 mutants encode constitutively active DEG/ENaC channels that are blocked by divalent cations

Ion channels of the DEG/ENaC family can induce neurodegeneration under conditions in which they become hyperactivated. The Caenorhabditis elegans DEG/ENaC channel MEC-4(d) encodes a mutant channel with a substitution in the pore domain that causes swelling and death of the six touch neurons in which it is expressed. Dominant mutations in the C. elegans DEG/ENaC channel subunit UNC-8 result in uncoordinated movement. Here we show that this unc-8 movement defect is correlated with the selective death of cholinergic motor neurons in the ventral nerve cord. Experiments in Xenopus laevis ooctyes confirm that these mutant proteins, UNC-8(G387E) and UNC-8(A586T), encode hyperactivated channels that are strongly inhibited by extracellular calcium and magnesium. Reduction of extracellular divalent cations exacerbates UNC-8(G387E) toxicity in oocytes. We suggest that inhibition by extracellular divalent cations limits UNC-8 toxicity and may contribute to the selective death of neurons that express UNC-8 in vivo.     

The role of hydrated divalent metal ions in the bridging of two anionic groups. An ab initio quantum chemical and molecular mechanics study of dimethyl phosphate and formate bridged by calcium and magnesium ions

Ab initio quantum chemical (Gaussian82) and molecular mechanics (AMBER2.0) computational techniques are employed to investigate the interaction of two anions (formate an dimethylphosphate) and a central divalent metal cation (magnesium or calcium). These systems are models for the essential GDP binding unit of the G-proteins (e.g., EF-Tu or the ras oncogene proteins) and for protein/phospholipid interactions, both of which are mediated by divalent metal cations. Various levels of hydration are utilized to examine coordination of differences between magnesium and calcium ions. Two different orientations of formate and dimethyl phosphate in direct ion contact with a magnesium ion and two waters of hydration were energy minimized with both quantum and molecular mechanics techniques. The structures and energy differences between the two orientations determined by either of the computational techniques are similar. Magnesium ion has a strong propensity to assume six coordination whereas calcium ion preferentially assumes a coordination greater than six. Likewise, water molecules attached to magnesium ion are held more rigidly than those of calcium ion, thus calcium ion is more accommodating in the exchange of water for negative ligands.     

Self-incompatibility in Papaver rhoeas activates nonspecific cation conductance permeable to Ca2+ and K+

Cellular responses rely on signaling. In plant cells, cytosolic free calcium is a major second messenger, and ion channels play a key role in mediating physiological responses. Self-incompatibility (SI) is an important genetically controlled mechanism to prevent self-fertilization. It uses interaction of matching S-determinants from the pistil and pollen to allow "self" recognition, which triggers rejection of incompatible pollen. In Papaver rhoeas, the S-determinants are PrsS and PrpS. PrsS is a small novel cysteine-rich protein; PrpS is a small novel transmembrane protein. Interaction of PrsS with incompatible pollen stimulates S-specific increases in cytosolic free calcium and alterations in the actin cytoskeleton, resulting in programmed cell death in incompatible but not compatible pollen. Here, we have used whole-cell patch clamping of pollen protoplasts to show that PrsS stimulates SI-specific activation of pollen grain plasma membrane conductance in incompatible but not compatible pollen grain protoplasts. The SI-activated conductance does not require voltage activation, but it is voltage sensitive. It is permeable to divalent cations (Ba(2+) ≥ Ca(2+) > Mg(2+)) and the monovalent ions K(+) and NH(4)(+) and is enhanced at voltages negative to -100 mV. The Ca(2+) conductance is blocked by La(3+) but not by verapamil; the K(+) currents are tetraethylammonium chloride insensitive and do not require Ca(2+). We propose that the SI-stimulated conductance may represent a nonspecific cation channel or possibly two conductances, permeable to monovalent and divalent cations. Our data provide insights into signal-response coupling involving a biologically important response. PrsS provides a rare example of a protein triggering alterations in ion channel activity.     

Cloning,Expression and Characterization of a Lipase Encoding Gene from Human Oral Metagenome

The human oral metagenomic DNA cloned into plasmid pUC19 was used to construct a DNA library in Escherichia coli. Functional screening of 40,000 metagenomic clones led to identification of a clone LIP2 that exhibited halo on tributyrin agar plate. Sequence analysis of LIP2 insert DNA revealed a 939 bp ORF (omlip1) which showed homology to lipase 1 of Acinetobacter junii SH205. The omlip1 ORF was cloned and expressed in E. coli BL21 (DE3) using pET expression system. The recombinant enzyme was purified to homogeneity and the biochemical properties were studied. The purified OMLip1 hydrolyzed p-nitrophenyl esters and triacylglycerol esters of medium and long chain fatty acids, indicating the enzyme is a true lipase. The purified protein exhibited a pH and temperature optima of 7 and 37 °C respectively. The lipase was found to be stable at pH range of 6–7 and at temperatures lower than 40 °C. Importantly, the enzyme activity was unaltered, by the presence or absence of many divalent cations. The metal ion insensitivity of OMLip1offers its potential use in industrial processes.     

Attachment of rat hepatocytes in vitro to substrata of serum protein, collagen, or concanavalin A.

Isolated rat hepatocytes attach to, and spread on, the surface of polystyrene tissue culture dishes in the presence of serum. The attachment is essentially complete in 30 min at 37 °C, whereas no attachment occurs at 0 °C. Dead (trypan blue-stainable) cells do not attach; hence the plating efficiency (percentage of cells attaching) is close to the percentage of intact cells in the hepatocyte suspension. Attachment in the presence of serum is relatively independent of pH, but requires divalent cations. Mg²⁺ stimulates attachment more effectively than Ca²⁺, and a combination of both cations gives maximal attachment. Cells do not attach readily to untreated dishes in the absence of serum, but attach to and spread on dishes precoated with adsorbed serum protein, concanavalin A (ConA), or a film of collagen. The attachment-promoting activity in serum is destroyed by acid treatment, by heating to 70 °C, and by protease treatment. It is therefore most probably a protein, which, like collagen and ConA, can bind to receptors on the hepatocyte surface.     

Biochemical analysis of a highly specific, pH stable xylanase gene identified from a bovine rumen-derived metagenomic library

A metagenomic library was generated using microbial DNA extracted from the rumen contents of a grass hay-fed dairy cow using a bacterial artificial chromosome-based vector system. Functional screening of the library identified a gene encoding a potent glycoside hydrolase, xyn10N18, localised within a xylanolytic gene cluster consisting of four open-reading frames (ORFs). The ORF, xyn10N18, encodes an endo-β-1,4-xylanase with a glycosyl hydrolase family 10 (GH10) catalytic domain, adopts a canonical α8/ß8-fold and possesses conserved catalytic glutamate residues typical of GH10 xylanases. Xyn10N18 exhibits optimal catalytic activity at 35 °C and pH 6.5 and was highly stable to pH changes retaining at least 85 % relative catalytic activity over a broad pH range (4.0–12.0). It retained 25 % of its relative activity at both low (4 °C) and high (55 °C) temperatures, however the stability of the enzyme rapidly decreased at temperatures of >40 °C. The specific activity of Xyn10N18 is enhanced by the divalent cations Mn²⁺ and Co²⁺ and is dramatically reduced by Hg²⁺ and Cu²⁺. Interestingly, EDTA had little effect on specific activity indicating that divalent cations do not function mechanistically. The enzyme was highly specific for xylan containing substrates and showed no catalytic activity against cellulose. Analysis of the hydrolysis products indicated that Xyn10N18 was an endoxylanase. Through a combination of structural modelling and in vitro enzyme characterisation this study provides an understanding of the mechanism and the substrate specificity of this enzyme serving as a starting point for directed evolution of Xyn10N18 and subsequent downstream use in industry.     

The guanine nucleotide-binding protein Gs activates a novel calcium transporter in Xenopus oocytes.

Calcium influx is an important aspect of receptor-mediated signal transduction, yet limited information is available regarding the pathways of calcium influx into nonexcitable cells. We show that treatment of oocytes from Xenopus laevis with cholera toxin, a potent activator of the guanine nucleotide-binding protein Gs, specifically stimulates a sustained inward whole cell flux of calcium through a novel membrane transporter. The calcium is distributed into a mobilizable pool. The flux is voltage-independent and is completely and specifically blocked by microinjection of oocytes with an antiserum directed against Gs alpha. The flux is not activated by treatment of the cells with forskolin or 8-bromo-cyclic adenosine monophosphate indicating that the effect of Gs alpha on the transporter occurs independently of adenylylcyclase activation. Transporter activity is insensitive to benzyl amiloride, does not require a sodium gradient, and is not stimulated by external calcium, indicating that it is not a sodium-calcium exchanger. The Gs-activated flux is dramatically potentiated by lanthanum ion and other trivalent cations but not by any of six divalent cations that were tested; all other known calcium channels and exchangers are, in contrast, potently blocked by lanthanum. The divalent cation cadmium inhibited transporter activity in a concentration-dependent manner. This novel calcium transporter may be important for receptor-mediated calcium influx in the oocyte and perhaps other cell types.     

Factors in the inactivation of postjunctional membrane receptors of frog skeletal muscle

Several factors which influence the rate of inactivation of muscle postjunctional membrane (PJM) receptors during the sustained application of carbamylcholine (CARB) have been studied by two methods. The rate of inactivation was increased by elevating the tonicity of the bathing medium, by increasing the CARB concentration, by raising the calcium ion concentration, and by substituting SO₄ ⁼ for Cl^- ions in the extracellular fluid. The relative effectiveness of calcium and other divalent cations in receptor inactivation was compared. In the absence of calcium, other divalent cations such as magnesium, strontium, or manganese were not efficient substitutes for calcium. In the presence of calcium, the addition of strontium or manganese ions accelerated the rate of receptor inactivation, but the addition of magnesium (up to 12 mM) inhibited this process. The inactivation of the membrane receptors in denervated muscle fibers was found to be similar to that in innervated muscle fibers. Various factors in PJM receptor inactivation are discussed. It is suggested that PJM receptor inactivation is influenced by the binding of calcium ions to sites on the internal surface of the PJM.     

Relief of Casein Inhibition of Bacillus stearothermophilus by Iron, Calcium, and Magnesium

Growth of Bacillus stearothermophilus strain NCA 1518 Smooth in Dextrose Tryptone Agar (DTA) was inhibited by sodium caseinate. Binding studies indicated that sodium caseinate, when present in DTA, had the capacity to effect an iron deficiency which could cause inhibition of growth. Additions of essential cations, iron (1 mM), calcium (5 mM), magnesium (10 mM), or hydrogen ion (pH 5.7), relieved inhibition. Responses to and interactions among these relief factors were analyzed statistically. Equations were fitted to the data and were used to estimate responses to all treatment combinations within the ranges tested. Results from these studies indicated that calcium, magnesium, and hydrogen ion acted by decreasing the binding capacity of the protein for iron, rendering this metal available for metabolic needs. Evidence was obtained that ferrous rather than ferric iron was the limiting factor in DTA containing sodium caseinate.