Characterization of the Hemolytic Properties of an Extract from Phaeocystis globosa Scherffel

Phaeocystis globosa Scherffel, an organism that causes harmful algal blooms, is a genus of the family Prymnesiophyta (or Haptophyta) with eurythermal and euryhaline characteristics. P. globosa has been confirmed to produce hemolytic substances, which are a mixture of liposaccharides. In the present study, the hemolytic properties of extract ofP. globosa are analyzed further. The effects of temperature, pH,different divalent cations, and membrane lipids on extract-induced hemolysis are discussed, as is the possible hemolytic mechanism. The results of the present study showed that the hemolytic activity of the extract was approximately 127.1 hemolytic units (HU)/L. The hemolytic reaction became fastest and a 50% decrease in absorbance was induced at 30 min at 37 ℃, and at pH 7.0; Hg2 was the strongest inhibitor of the hemolysis compared with the other divalent cations and many membrane lipids, except for phosphatidic acid, inhibited the hemolytic activity to different degrees. These results suggest that the toxin may make pores in the surface of red blood cells and that Hg2 either combines with the hemolysin or closes the pores,hence inhibiting its further hemolytic reaction. The toxin probably has no specific membrane receptor in the red blood cell membrane.     

Purification and characterization of adenine phosphoribosyltransferase from Arabidopsis thaliana

Adenine phosphoribosyltransferase (APRT; EC 2. 4,2. 7) from Arabidopsis thaliana was purified approximately 3800-fold, to apparent homogeneity. The purification procedure involved subjecting a leaf extract to heat denaturation, (NH₄)₂SO₄ precipitation, Sephadex G-25 salt separation, ultracentrifugation and liquid chromatography on Diethylaminoethyl Sephacel, Phenyl Sepharose CL-4B, Blue Sepharose CL-6B and adenosine 5'-monophosphate-Agarose. The purified APRT was a homodimer of approximately 54 kDa and it had a specific activity of approximately 300 μmol (mg total protein)^-1 min^-1. Under standard assay conditions, the temperature optimum for APRT activity was 65°C and the pH optimum was temperature dependent. High enzyme activity was dependent upon the presence of divalent cations (Mn²⁺ or Mg²⁺). In the presence of MnCl₂₊ other divalent cations (Mg²⁺, Ca²⁺, Ba²⁺, Hg²⁺ and Cd²⁺) inhibited the APRT reaction. Kinetic studies indicated that 5-phosphoribose-1-pyrophosphate (PRPP) caused substrate inhibition whereas adenine did not. The K_m for adenine was 4.5±1.5 μ M , the K_m for PRPP was 0.29±0.06 m M and the K_i for PRPP was 1.96±0.45 m M . Assays using radiolabelled cytokinins showed that purified APRT can also catalyze the phosphoribosylation of isopentenyladenine and benzyladenine. The K_m for benzyladenine was approximately 0.73±0.06 m M     

Cations Affect [3H]Mazindol and [3H]WIN 35,428 Binding to the Human Dopamine Transporter in a Similar Fashion

Abstract: The present study addresses the possibility that there are different cocaine-related and mazindol-related binding domains on the dopamine transporter (DAT) that show differential sensitivity to cations. The effects of Zn²⁺, Mg²⁺, Hg²⁺, Li⁺, K⁺, and Na⁺ were assessed on the binding of [³H]mazindol and [³H]WIN 35,428 to the human (h) DAT expressed in C6 glioma cells under identical conditions for intact cell and membrane assays. The latter were performed at both 0 and 21°C. Zn²⁺ (30–100 µ M ) stimulated binding of both radioligands to membranes, with a relatively smaller effect for [³H]mazindol; Mg²⁺ (0.1–100 µ M ) had no effect; Hg²⁺ at ∼3 µ M stimulated binding to membranes, with a relatively smaller effect for [³H]mazindol than [³H]WIN 35,428 at 0°C, and at 30–100 µ M inhibited both intact cell and membrane binding; Li⁺ and K⁺ substitution (30–100 m M ) inhibited binding to membranes more severely than to intact cells; and Na⁺ substitution was strongly stimulatory. With only a few exceptions, the patterns of ion effects were remarkably similar for both radioligands at both 0 and 21°C, suggesting the involvement of common binding domains on the hDAT impacted similarly by cations. Therefore, if there are different binding domains for WIN 35,428 and mazindol, these are not affected differentially by the cations studied in the present experiments, except for the stimulatory effect of Zn²⁺ at 0 and 21°C and Hg²⁺ at 0°C.     

Effects of Microinjected Cations on the Early Event of Fertilization in the Medaka Egg

Effects of microinjected cations on the early events of fertilization were examined using eggs of Oryzias latipes . Microinjection of either Ca²⁺, Ba²⁺ or Sr²⁺ into the thin cortical cytoplasm induced breakdown of cortical alveoli (vesicles) (CABD) under Ca-Mg-free conditions, but microinjection of Mg²⁺, Mn²⁺ or Co²⁺ prevented CABD at the injected region when the eggs were inseminated in regular saline. Under Ca-Mg-free conditions, CABD could also be induced by microinjection of various solutions (NaCl, choline chloride, sucrose, pH buffer) without any divalent cations or ionophore A23187. Ca²⁺ microinjected into the cortical cytoplasm did not play a role in sperm penetration. Upon microinjection with either Ca²⁺, Mg²⁺ or K⁺, the resting membrane potential leakage was transiently observed. However, depolarization of the membrane followed by slow hyperpolarization was observed only upon microinjection of Ca²⁺. From these experiments, it was inferred that microinjected divalent cations such as Ca²⁺, Ba²⁺ or Sr²⁺ do not act directly upon the cortical alveolus membrane, but trigger the induction of CABD via depolarization of the membrne and increase in intracellular Ca²⁺.     

Enhancement of Tryptophan Uptake by Divalent Cations in the Absence of Sodium Ions

Abstract: Nations were found to inhibit the uptake of L-tryptophan into synaptosomes with a shallow dose-response curve. Almost maximal inhibition was obtained with 10 mM-Na⁺. The divalent cations Ca²⁺ and Mg²⁺ were shown to be responsible for the increased uptake of L-tryptophan in the absence of Na⁺ ions. Other divalent cations also promoted tryptophan uptake under this condition (Ca²⁺ < Mg²⁺ < Mn²⁺ < Fe²⁺ < Zn²⁺ < Cu²⁺). It was concluded that monovalent chelate complexes were responsible for this enhancing effect. The measured L-tryptophan uptake was the net product of membrane bound and unbound tryptophan. Both bound and unbound tryptophan were increased in the presence of divalent cations. If no divalent cations were added to the incubation medium, Na⁺ ions decreased the unbound tryptophan but were without effect on bound tryptophan. Under these circumstances D-tryptophan had no effect on binding of the L-isomer and affected the transport of 1.-tryptophan only at very high does (100 x conc. L-tryptophan). These results suggest that I -tryptophan binds to a stereospecific transport carrier located in the synaptosomal membrane and that Na⁺ ions prevent the translocation of this carrier amino acid complex from the outer to the inner site of the neuronal membrane.     

Properties of NADP+-malic enzyme from pod walls of chickpea (Cicer arietinum)

NADP⁺-malic enzyme ( l -malate: NADP⁺ oxidoreductase, decarboxylating EC 1.1.1.40) from pod walls of chickpea was purified 51-fold by ammonium sulphate fractionation, DEAE- cellulose chromatography and gel filtration through Sepharose 4B. The purified enzyme required a divalent cation, either Mn²⁺ or Mg²⁺, for its activity. K_m values at pH 7.8 for malate, NADP⁺ and Mn²⁺ were 4.0, 0.031 and 0.71 m M , respectively. Mn²⁺-dependent activity was inhibited by heavy metal ions such as Cd²⁺, Zn²⁺, Hg²⁺, and to a lesser extent by Pb²⁺ and Al³⁺. Among the organic acids examined, sodium salts of oxalate and oxaloacetate were inhibitory. Kinetics of the reaction mechanism showed sequential binding of malate and NADP⁺ to the enzyme. Products of reaction, viz. pyruvate, bicarbonate and NADPH, inhibited the enzyme activity. At limiting concentrations of NADP⁺, pyruvate and bicarbonate induced a positive cooperative effect by malate. It is proposed that the activity of NADP⁺-malic enzyme is controlled by intracellular concentrations of substrates and products.     

Requirement of Extracellular Calcium Ions for the Early Fertilization Events in the Medaka Egg

The requirement for calcium and the change in calcium content in eggs of Oryzias iatipes during the cortical reaction and sperm penetration were examined. Naked eggs failed to exhibit the cortical reaction upon insemination under Ca Mg-free conditions. These eggs exhibited the cortical reaction by reinsemination in the presence of extracellular Ca²⁺. The effect of extracellular Ca²⁺ on sperm penetration could be replaced by one of several divalent cations in the external medium. Unlike the cortical reaction, sperm penetration failed to be induced by microinjection to increase intracellular Ca²⁺. Verapamil significantly reduced the action of extracellular Ca²⁺ or Ba²⁺ of divalent cations examined in fertilization, while TEA and TTX had no effect on fertilization in the presence of these cations. No ⁴⁵Ca uptake into the egg proper was recognized before completion of the cortical reaction. These observations suggest that extracellular divalent cations are indispensable for sperm stimulation of the egg and its penetration into the egg, for which an influx of Ca²⁺ from the external medium is not required.     

Divalent cation inhibition of barley root plasma membrane-bound Ca2+-ATPase activity and its reversal by monovalent cations

The inhibitory action of divalent cations on the Ca²⁺-ATPase activity of a plasma membrane-rich microsome fraction isolated from the roots of barley ( Hordeum vulgare L. cv. Conquest) was investigated. Using electron paramagnetic resonance spectroscopy to measure cation-induced changes in membrane lipid properties, it was demonstrated that certain divalent cations (Ca²⁺, Cd²⁺, UO²⁺₂) inhibit the Ca²⁺ ATP-ase by restriction of lipid polar head group mobility and not by alteration of membrane surface potential. Monovalent cations which stimulate the Ca²⁺-ATPase of barley roots (Na⁺, K⁺, ethanolamine HCl) can also reverse the Ca²⁺-ATPase inhibition by Cd²⁺. The degree of Na⁺ reversal of Cd²⁺-induced Ca²⁺-ATPase inhibition was influenced by the nature of the anion.     

Possible involvement of red pigments in defense against mercury in Pseudomonas K-62

Abstract To study the physiological role of the red pigments in soil strain Pseudomonas K-62, we isolated a red pigment-deficient white mutant from the soil strain by treatment with mitomycin C and compared the phenotypic properties of the mutant and parent strain. The red pigments, which were classified as one of carotenoids based on their physicochemical properties, were separated into two groups, designated pigment A and B respectively on NH-Chromatorex HPLC.The crude pigments and pigment B which could react with Hg²⁺ in the wild-type Pseudomonas K-62 and its mercury-resistant plasmid-deficient strain were enhanced by the addition of Hg²⁺. The white mutant thus obtained showed a greater sensitivity to Hg²⁺ than the wild-type reddish strain despite containing the resistant plasmids. The major component in pigment B was identified by mass spectrometric analysis as 1-hydroxy-1-methoxy-1,2, 1',2',7',8'-hexahydro-ψ,ψ-caroten-4-one, a carotenoid monoketone. These results suggested that red pigments, especially pigment B, may account, at least partially, for defense against Hg²⁺ in the bacterial environments.     

Chitinase production by Streptomyces viridificans: its potential in fungal cell wall lysis

R. GUPTA, R.K. SAXENA, P. CHATURVEDI AND J.S. VIRDI. 1995. Streptomyces viridificans was found to be a good chitinase producer among nine species of Streptomyces screened. Minimum levels of constitutive enzyme were observed with both simple and complex carbon substrate. Arabinose doubled the enzyme production amongst the various pentoses and hexoses used with chitin. However, with glucose end-product inhibition and catabolite repression were observed. The enzyme tolerated a wide range of temperature (30–55°C) and pH (3–7˙5). Among various divalent cations Mn²⁺ and Hg²⁺ completely inhibited the purified enzyme while β-mercaptoethanol stimulated its activity. Crude and purified enzyme had potential for cell wall lysis of many fungal pathogens tested.     

Stimulation of proton extrusion by K+ and divalent cations (Ni2+, Co2+ Zn2+) in maize root segments

Entry of the divalent cations Ni²⁺, Co²⁺ and Zn²⁺ into cells of maize ( Zea mays L. cv. Dekalb XL 85) root tissue is accompanied by an acidification of the incubation medium, a decrease in both the pH of the cell sap and the level of malate in the cells, and by an inhibition of dark fixation of CO₂. K⁺, on the contrary, induces only a very low acidification of the incubation medium, does not change either the pH of the cell sap or the malate level in the cells, and induces an increase in CO₂ dark fixation. Different mechanisms are postulated for the stimulation of proton extrusion by divalent cations and K⁺.     

DIVALENT CATION-ACTIVATED ECTO-NUCLEOSIDE TRIPHOSPHATASE ACTIVITY OF NERVOUS SYSTEM CELLS IN TISSUE CULTURE

Abstract— Intact neuroblastoma and glial cells in monolayer culture hydrolysed ATP added to their medium. Evidence is presented that ATP is cleaved outside of the permeability barrier of the plasma membrane and the product is liberated in the extracellular medium, i.e. the enzyme is an ecto-enzyme. Divalent cations such as Mg²⁺, Ca²⁺, Mn²⁺ and Co²⁺ activate the enzyme. In neuroblastoma cells, Ca²⁺ is the preferential cation for activation; Mg²⁺ in glial cells. Substrate specificity was very low when different nucleoside-5'-triphosphates were examined. Competition studies have revealed that all of the nucleoside triphosphates are hydrolysed by the same enzyme: divalent cation-activated ecto-nucleoside-5'-triphosphate phosphohydrolase.
Developmental pattern of the enzyme in several lines was established. The role of enzyme in the transport of divalent cations across the plasma membrane and/or in the physical properties of the membrane is suggested.     

Characterization of mitochondrial glutamate dehydrogenase from dark‐grown soybean seedlings

Purified preparations of NAD(H)‐glutamate dehydrogenase (GDH, EC 1.4.1.2.) were assayed to determine the effects of mono‐ and divalent cations, nucleotides and select carbon compounds on NAD(H)‐dependent GDH activity. The amination reaction was stimulated 2‐ to 17‐fold by divalent cations (Ca²⁺ > Cd²⁺ > Co²⁺ > Mg²⁺ > Mn²⁺ > Zn²⁺ between 1 and 1000 µ M ), but the reaction was unaffected by monovalent cations (Na ⁺ and K ⁺). The amination reaction was most responsive to changes in Ca²⁺ in a NADH‐dependent manner. The addition of EDTA or EGTA nullified the stimulatory effects of Ca²⁺. Calmodulin alone or in combination with calmodulin antagonists did not affect the amination reaction. Divalent cations (at 1 m M ) inhibited the rate of the deamination reaction by 15 to 25%, while monovalent cations had no effect. ATP inhibited the amination reaction by 10 to 60%, while ADP had little or no effect. ATP or ADP decreased the rate of the deamination reaction 23 to 60 or 20 to 38%, respectively. Many tricarboxylic acid cycle intermediates inhibited the amination reaction, 20 to 50% of the inhibition could be attributed to the chelating capacity of intermediates. Conversely, most of the carbon sources tested did not affect the deamination reaction, the only appreciable differences were increases in activity with sucrose (21%) and glucose (41%) and a decrease in activity with pyruvate (34%). Inhibitors of sulfhydryl groups were used to examine the importance of reduced thiol groups in the amination or deamination reactions. The amination was not dependent on reduced thiol groups, whereas the deamination reaction was dependent on reduced thiol groups.     

Apoplastic pH and inorganic ion levels in tomato fruit: A potential means for regulation of cell wall metabolism during ripening

Apoplastic pH and ionic conditions exert strong influence on cell wall metabolism of many plant tissues; however, the nature of the apoplastic environment of ripening fruit has been the subject of relatively few studies. In this report, a pressure-bomb technique was used to extract apoplastic fluid from tomato fruit ( Lycopersicon esculentum Mill.) pericarp at several developmental stages. pH and the levels of K⁺, Na⁺, Ca²⁺, Mg²⁺, Cl⁻ and P were determined and compared with the values for the bulk pericarp and locule tissues. The pH of the apoplastic fluid from pericarp tissue decreased from 6.7 in immature and mature-green fruits to 4.4 in fully-ripe fruit. During the same period, the K⁺ concentration increased from 13 to 37 m M . The levels of Na⁺ and divalent cations did not change, whereas the anions P and Cl⁻ increased in ripe fruit. Ca²⁺ levels remained relatively constant during ripening at 4–5 m M , concentrations that effectively limit pectin solubilization. The electrical conductivity of the apoplastic liquid increased 3-fold during ripening, whereas osmotically active solutes increased 2-fold. Pressure-treated fruit retained the capacity to ripen. The decline in apoplastic pH and increase in ionic strength during tomato fruit ripening may regulate the activity of cell wall hydrolases. The potential role of apoplastic changes in fruit ripening and softening is discussed.     

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.     

p-NITROPHENYL PHOSPHATASES OF A MEMBRANE FRACTION FROM BOVINE CEREBRAL CORTEX

Abstract— Three different types of p -nitrophenyl phosphatases (NPPases) were solubilized by deoxycholate treatment from a membrane fraction of bovine cerebral cortex, and their characteristics were determined. Of these three NPPases (acid, Mg²⁺-activated, and K⁺, Mg²⁺-activated), only K-Mg NPPase was stimulated about two-fold by phospholipid and was inhibited by unsaturated neutral lipids and fatty acids. Unlike Na⁺-K⁺-Mg^a+-activated ATPase, the enzyme did not absolutely require phospholipid for its activity, but was similarly thermolabile and was protected by phospholipid from thermal inactivation. Acid NPPase was separable from the other two NPPases by ammonium sulphate fractionation, and partly solubilized by dialysis against ATP-mercaptoethanol solution. Hg²⁺ inhibited equally all three NPPases, but Ca²⁺ inhibited only Mg and K-Mg NPPases. Ouabain was effective on K-Mg NPPase alone.     

Effect of low growth temperature on coupling between electron transport and proton flux in Vicia faba thylakoids

Coupling between electron transport and proton flux has been compared in chloroplasts from Vicia faba (cv. Windsor) plants grown at 20 and 5°C. Proton uptake by warm-grown thylakoids was sensitive to external pH and stimulated by micromolar adenine nucleotide above pH 7.0. Electron transport was modulated by pH, adenine nucleotide and energy transfer inhibitors (triphenyltin and Hg²⁺). By contrast, proton uptake by cold-grown thylakoids was generally lower and was insensitive to micromolar ATP. The rate of non-phosphorylating electron flow in cold-grown thylakoids was relatively insensitive to pH and Hg²⁺ and was not modulated by adenine nucleotides or triphenyltin. Stimulation of electron transport by phosphorylating conditions in cold-grown thylakoids was generally lower and insensitive to pH. It is concluded that the control of proton efflux through CF₀-CF₁ differs in thylakoids of V. faba grown at warm and cold temperatures.     

Characterization of cyclic nucleotide phosphodiesterase activity in Phaseolus vulgaris

Cyclic nucleotide phosphodiesterase (3',5'-cyclic nucleotide nucleotidohydrolase, EC 3.1.4.17) activity isolated from Phaseolus vulgaris L. cv. Limberg seedlings was partially purified and characterized by fractional (NH₄)₂SO₄ precipitation, DEAE-cellulose chromatography, chromatography on 3',5'-cAMP-agarose, gel permeation chromatography and chromatofocusing. A crude enzyme preparation, a 30–65% (NH₄)₂SO₄ pellet, showed an acidic pH optimum. The enzyme activity was stimulated by imidazole and divalent cations such as Ca²⁺, Mg²⁺ and Mn²⁺, whereas NaF, PP_i and Fe³⁺ were inhibitory. Isobutylmethylxanthine had no significant effect on the plant enzyme. An M_I of 42 000 was estimated by gel permeation high performance liquid chromatography. By chromatography on 3',5'-cAMP-agarose a phosphodiesterase was resolved that produced 5'-AMP as sole reaction product.     

Characterization of sperm motility in sea bass: the effect of heavy metals and physicochemical variables on sperm motility

Computer assisted sperm analysis (CASA) was used to characterize the motility of sea bass Dicentrarchus labrax spermatozoa and to study the effect of several physicochemical variables and heavy metals on sperm swimming performance. Duration of sperm motility in sea bass was very short (<50 s). During the first 20 s all the motility variables measured remained approximately constant, the velocity and linearity of the movement being maximum during this period, while both variables decreased sharply later. While slight variations in pH did not significantly modify sperm swimming performance, changes in osmolality affected all the measured motility variables. Two of the heavy metals tested, Cu²⁺ and Pb²⁺, did not affect sperm motility when the activating media contained up to 100 ppm of the metal salts. In contrast, Hg²⁺ modified the morphology of post-swimming spermatozoa at 0·4–1 ppm (sperm dilution rate 1:39) and completely arrested sperm motility at concentrations as low as 0·1 ppm (sperm dilution rate 1:2500). Assuming a covalent binding to sperm cells, this revealed a finite number of c. 10 million Hg²⁺ binding sites per spermatozoon. Complementary results using demembranated spermatozoa suggested that the main target of HgCl₂ would be located in the plasma membrane and that HgCl₂ would inhibit water channels, hence preventing sperm motility.     

Effect of divalent cations on permeabilizer-induced lysozyme lysis of Pseudomonas aeruginosa

The presence of divalent (Mg²⁺) ions greatly reduced the lysis of Pseudomonas aeruginosa strain G48 in a system at pH 7·8 or 9·0 consisting of ethylenediamine tetraacetic acid (EDTA), lysozyme and tris. Similar reductions in lysis occurred when EDTA was replaced by nitrilotriacetic acid, sodium citrate or sodium polyphosphate. The effect depended on the cation concentration. Mg²⁺ may replace cations removed from the outer membrane, or may effectively remove the permeabilizer from the system. The results suggest that the permeabilizing activity associated with these agents against this organism has a common basis in affecting the outer membrane.