Divalent cation binding to phospholipid vesicles. Dependence on temperature and lipid fluidity

Mn²⁺ binding to vesicles prepared from several different species of anionic phospholipids was determined as a function of temperature by electron paramagnetic resonance (EPR). The Mn²⁺ affinities of phosphatidylserine, cardiolipin and egg yolk phosphatidylglycerol all increased monitonically with temperature.Vesicles prepared from hydrogenated and natural (bovine) phosphatidylserine were monitored with respect to hydrocarbon chain fluidity as well as Mn²⁺ binding. Contrary to expectations based on surface potential considerations, the affinity of phosphatidylserine for divalent cations was apparently not lowered in going from the gel state to the liquid crystalline state of the bilayer. The results are instead consistent with an enhancement in cation affinity with increased lipid fluidity.Dipalmitoyl phosphatidylglycerol vesicle fluidity and Mn²⁺ binding were also studied with EPR. A large reduction in the measured Mn²⁺ affinity accompanied melting of the phospholipid, but observed hysteresis in the temperature dependence of the binding render uncertain any simple explanation based on changes in surface potential. Supplementary light scattering data indicated that vesicle aggregation was involved in the hysteresis phenomena.     

Effects of Manganese Deficiency on Spectral Characteristics and Oxygen Evolution in Maize Chloroplasts

The effects of Mn²⁺ deficiency on light absorption, transmission, and oxygen evolution of maize chloroplasts were investigated by spectral methods. Several effects of Mn²⁺ deficiency were observed: (1) the skeleton of pigment protein complexes and oxygen-evolving center and the combination between pigment and protein were damaged; (2) the light absorption of chloroplasts was obviously decreased; (3) the energy transfer among amino acids within PS II protein–pigment complex and decreased energy transport from tyrosine residue to chlorophyll a and from chlorophyll b and carotenoid to chlorophyll a were inhibited; (4) the oxygen-evolving of chloroplast was significantly inhibited. However, Mn²⁺ addition decreased the damage of light absorption, transmission, and oxygen evolution of maize chloroplasts caused by Mn²⁺ deficiency.     

The binding of Mn2+ and ADP to myosin

The metal ion requirement of myosin-ADP binding was investigated by use of Mn²⁺. Mn²⁺ binds to two sets of noninteracting sites on myosin which are characterized by affinity constants of 10⁶ and 10³, M⁻¹ at 0.016 M KCl concentration. The maximum number of sites is 2 for the high affinity and 20–25 for the low affinity set. Binding of Mn²⁺ to the high affinity sites increases the affinity of ADP binding to myosin. F-actin inhibits ADP binding (Kiely, B., and Martonosi, A., Biochim. Biophys. Acta 172: 158–170 [1969]), but even at F-actin concentrations much higher than that required to saturate the actin binding sites of myosin or its proteolytic fragments, significant ADP binding remained. The actin insensitive portion of ADP binding was inhibited by 10⁻⁴ M inorganic pyrophosphate or ATP. The results are discussed on the basis of a model in which actin and ADP bind to myosin at distinct but interacting sites.     

Activation of Electron Donation from Hydrogen Peroxide by Manganese in Non-oxygen evolving Photosystem II Particles

Analysis of the activation of H₂O₂-supported 2,6-dichloroindophenol(DCIP) photoreduction by MnCl₂ showed two Mn²⁺-binding sitesin non-oxygen evolving PS II particles, with large (0.4IM) andsmall (0.04 µm) K_m values for Mn²⁺. Photoreduction throughthe high-affinity Mn²⁺.-binding site was inhibited by treatmentwith H₂O₂. (Received April 20, 1987; Accepted July 13, 1987)     

Effects of bicarbonate ion on serotonin binding to rat frontal cortex membranes

Several factors are known to regulate ligand binding to 5-hydroxytryptamine (5-HT) receptors. In the present experiments we have investigated the mechanism by which bicarbonate ion modify central 5-HT receptor sensitivity in rats. Mn²⁺ (10⁻⁶–10⁻³M) increased specific [³H]5-HT binding to 5-HT₁ receptor sites (+60–70%), this effect being further enhanced by the addition of HCO⁻₃ (+300–400%), while the binding of [³H]spiperone binding to 5-HT₂ receptor sites was not affected by Mn²⁺ and HCO⁻₃. The effect of other divalent cations, Mg²⁺, Cu²⁺, Ca²⁺ and Fe²⁺, however, were not enhanced by the addition of HCO⁻₃. Scatchard analysis indicated that the effect of bicarbonate ion was associated with increase in the number of high affinity binding sites and appearance of low affinity binding sites. This effect of bicarbonate ion was characterized by decreased dissociation rate of the specific binding, was temperature-dependent, reduced by N-ethylmaleimide and iodoacetamide, and was completely inhibited by ascorbate, dithiothreitol and 2-mercaptoethanol. The effect was not influenced by GTP or GppNHp but it was significantly inhibited by ATP. Pretreatment of membranes with Triton X-100 (0.1%) increased the effect of bicarbonate ion. From these results, it is suggested that bicarbonate ion specifically interacts with Mn²⁺ and selectively increases [³H]5-HT binding.     

Removal of Mn from spinach chloroplasts by sodium cyanide and the binding of Mn2+ to Mn-depleted chloroplasts.

Manganese and copper were released from spinach chloroplasts by NaCN-treatment, though iron was not affected. The Hill reaction activity was also inhibited by this treatment, but was partially recovered by the addition of either Mn2+ or Cu2+, but not of Fe3+. The interaction of Mn2+ with manganese-depleted chloroplasts by NaCN-treatment was studied using 54Mn2+. A Scatchard plot shows the high and low affinity binding sites of Mn2+ on NaCN-treated chloroplast membrane; high affinity binding being specific for NaCN-treated chloroplast with a binding constant, KH, of 1.9 X 10(5) M-1, and a maximum binding number, NH, of 0.0016 g-atom per mole of chlorophyll. The low binding site was also found on untreated chloroplasts; its binding constant, KL, being 1.2 X 10(4) M-1, and its maximum binding number, NL, of 0.0112 g-atom per mole oc chlorophyll at pH 8.2 NH was proportional to the degree of the removal of Mn by NaCN-treatment and was constant at pH 4--9. NL markedly increased at a high pH with a midpoint of pH 7.9 indicating the exposure of a new, similar binding site. Light illumination partially inhibited the binding of Mn2+. Within 1 min in the dark the binding reaction reached equilibrium in the absence of pyrophosphate, however, 20 min were required to transform into pyrophosphate-resistant form. The pH dependence of the binding of Mn2+ with pKa 7.2 and the ineffectiveness of p-chloromercuribenzoate suggest the possible ligand of Mn2+ is the imidazole nitrogen of the histidine residue.     

Requirement of Manganese for Electron Donation of Hydrogen Peroxide in Photosystem II Reaction Center Complex

Mn²⁺ was required for the electron donating reaction from H₂O₂,but not for that from diphenylcarbazide (DPC), in the PS IIreaction center complex which was prepared from spinach chloroplastsby Triton X-100 extraction. The reaction center complex showeda high activity of 2,6-dichloroindophenol (DCIP) photoreductionin the presence of DPC, but a low activity with H₂O₂. The H₂O₂-supportedDCIP photoreduction was suppressed by EDTA and enhanced by asmall amount of Mn²⁺. Ca²⁺ and Mg²⁺ could not replace Mn²⁺.The activation by Mn²⁺ and its binding showed two binding sitesof Mn²⁺ in the reaction center complex, with high (1.5?10⁷ M^–1)and low (1 ? 10⁶ M^–1) binding constants. (Received November 8, 1986; Accepted April 10, 1987)     

Requirement of divalent cations for photoactivation of the laten water-oxidation system in intact chloroplasts from flashed leaves

The effects of divalent cations on photoactivation of the latent water-oxidation system in intact chloroplasts isolated from wheat (Triticum aestivum L.) leaves grown under intermittent flash illumination were investigated by using A23187, an ionophore for divalent cations, and the following results were obtained. (a) Photoactivation in the intact chloroplasts was inhibited by A23187, but was restored on addition of a low concentration of Mn²⁺ (10 μM). (b) A high concentration of Mn²⁺ (70 μM) was inhibitory, in contrast, for photoactivation, but the inhibition was restored by the coexistence of a suitable concentration of Ca²⁺ (5 mM). (c) The Ca²⁺-dependent restoration was inhibited by a high concentration of Mg²⁺ or Sr²⁺, but the inhibition was restored by the coexistence of Ca²⁺. (d) Kinetic analyses of these competitive effects between divalent cations revealed that: (i) High concentration of Ca²⁺ inhibits photoactivation in competition with Mn²⁺. (ii) High concentration of Mn²⁺ inhibits photoactivation in competition with Ca²⁺. (iii) High concentration of Mg²⁺ affects photoactivation by inhibiting Ca²⁺-dependent restoration in competition with Ca²⁺. Based on these results, we propose that the latent water-oxidation center has two binding sites, each specific for Mn²⁺ and Ca²⁺, and that photoactivation takes place in the center having both Mn²⁺ and Ca²⁺ on their respective binding sites.     

Comparative study of the reactivation of oxygen evolution in chloroplasts inhibited by various treatments

Reactivation of photosynthetic oxygen-evolution was investigatedwith chloroplasts inhibited by 0.8 M Tris-, 0.8 M Tris-20% acetone-,0.8 M KCl-, 0.5 M NaClO₄- or 1 mM NH₂OH-washing, and with heat-treatedor aged chloroplasts. These chloroplasts restored oxygen evolvingactivity by two successive treatments; incubation of chloroplastswith reduced DPIP, then with Mn^2$, Ca^2$, dithiothreitol andbovine serum albumin under weak illumination (light-reactivation). Some factors required for light-reactivation could be omitteddepending on the inhibition treatment. For example, Mn^2$, Ca^2$and dithiothreitol were not necessary for (1 mM NH₂OH-STN (pH7.0)-washed)-DPIP-treated chloroplasts, and dithiothreitol for(Tris-acetone (pH 8.4)-washed)-DPIP-treated chloroplasts. Uncouplers, such as atebrin, CCCP, DCCD and NH₄Cl, inhibitedthe lightreactivation. The Mn and Ca contents of the chloroplasts were determined withinhibited and DPIP-treated chloroplasts. The Mn content of thechloroplasts tended to decrease with increasing pH of the washingmedium for inhibition. The Ca content decreased when chloroplastswere washed with 0.8 M KCl. (Received November 22, 1974; )     

Characterization of homologous divalent metal ion binding sites of vertebrate and molluscan myosins using electron paramagnetic resonance spectroscopy.

The binding of Ca²⁺, Mg²⁺ and Mn²⁺ to myosins from rabbit skeletal muscle, scallop striated adductor muscle and clam adductor muscle has been investigated. All three myosins bind two moles of divalent metal ion non-specifically and with high affinity (Mn²⁺ > Ca²⁺ > Mg²⁺). In addition, the molluscan myosins bind about a further two moles of Ca²⁺ specifically. Although rabbit myosin binds some Ca²⁺ in the presence of an excess of free Mg²⁺, this binding occurs at the nonspecific sites and should not be taken as evidence for a myosin-linked regulatory system of the type found in molluscan muscles. If such a system exists in vertebrate skeletal muscle, the homologous Ca²⁺-specific sites must be lost during the early stages of the myosin preparation.The characteristic electron paramagnetic resonance spectrum of the bound Mn²⁺ was utilized to confirm the homology of the non-specific sites in vertebrate and molluscan myosins. The sites are located on the “regulatory” class of light chain. Mn²⁺ bound to scallop myosin has a broad electron paramagnetic resonance spectrum, in contrast to the well-resolved spectra that it gives when bound to many other myosin species. This situation was exploited to identify homologous nonspecific, divalent metal-ion sites on the regulatory light chains from a variety of muscle types, including frog skeletal, rabbit cardiac, chicken gizzard and molluscan adductor muscles. When these light chains are combined with desensitized scallop myofibrils the electron paramagnetic resonance spectra of Mn²⁺ bound to the resultant hybrids are dominated by the signal from the non-specific site of the foreign regulatory light chain.     

Effects of manganese, calcium, dithiothreitol and bovine serum albumin on the light-reactivation of Tris-acetone-washed chloroplasts

Oxygen-evolution activity of spinach chloroplasts was investigatedby washing chloroplasts with 0.8M Tris buffer containing 20%acetone. This inactivitation was easily removed by two successivetreatments, dark- and light-reactivations. The first treatmentwas dark-reactivation step, rewashing inactivated chloroplastswith reduced DPIP (DPIP treatment). The second one was a light-reactivatedchloroplasts with incubating chloroplasts with Mn²⁺, Ca²⁺, dithiothreitoland bovine serum albumin under ilumination. Both light- and dark-reactivation treatments were required toregain oxygen-evolution activity of Tris-acetone-washed chloroplasts,which is characteristic of such chloroplasts. However, in Tris-washedchloroplasts considerable activity was recovered by dark-reactivationalone. Manganese and calcium contents of Tris-acetone-washed chloroplastswere compared with those of chloroplasts obtained by other preparations. Tris-acetone washing was presumed to inhibit the oxygen-evolutionsite of Photo-systetm II by affecting Mn, Ca and other substancesin chloroplasts. The inhibition site was estimated from a changein fluorescence yield of chlorophyll and the effect of artificialelectron donor specific for Photosystem II on NADP photoreductionactivity. (Received August 20, 1973; )     

Fatty acid synthesis by spinach chloroplasts I. Property of fatty acid synthesis from acetate

Intact chloroplasts (about 70% Class I chloroplasts) isolatedfrom spinach leaves incorporated 150 nmoles of [1-¹⁴C] acetateinto fatty acids per mg chlorophyll in 1 hr at pH 8.3, 25°Cand 25,000 lux. On electron and phase-contrast microscopiescombined with hypotonic treatment of chloroplasts, this syntheticactivity was shown to be proportional to the percentage of ClassI chloroplasts in the preparation. Light was necessary for thesynthesis, the activity in the complete reaction mixture inthe dark being only 2% of that in the light. The synthetic activityincreased with increasing intensities of light to reach saturationat 6,000 lux. CoA and ATP were most effective as cofactors,HCO₃^–, HPO₄^2–, Mg^2$ and Mn^2$ were less effective.ATP could be replaced by ADP in the presence of Pi, suggestingpossible supply of ATP by photophosphorylation. Omission ofthe NADPH-generation system and NADH did not affect the synthesis,indicating sufficient provision of endogenous NADPH and NADHin intact chloroplasts under light. Addition of DTE did notcause recovery of the synthetic activity of intact chloroplastsin the dark. ¹ Present address: Radioisotope Centre, University of Tokyo,Yayoi, Bunkyo, Tokyo 113, Japan. (Received August 26, 1974; )     

Metal ion induced conformational changes in concanavalin A: evidence for saccharide binding to one metal free structure.

The addition of Mn²⁺, Zn²⁺, Co²⁺, Ca²⁺ or Pb²⁺ to apo-concanavalin A results in a slow conformational conversion of the protein to the active saccharide binding form. The rates of conversion are dependent upon the sample pH and identity of the ions which occupy the native transition metal and calcium ion sites yet the affinity of each metalloform for the fluorescent sugar, 4-methylumbelliferyl-α-D-mannopyranoside, is independent of these same parameters (above pH 5.6). EDTA quickly removes all metal ions from the active Mn²⁺ or Co²⁺-concanavalin A samples leaving a metastable metal free structure which retains its high saccharide affinity for several hours at room temperature. This form of apo-concanavalin A and the metallized derivatives have equally high saccharide binding affinities in 1M NaCL but the former dramatically loses its sugar affinity as the ionic strength is lowered.     

Studies on cytochromes in photosynthetic electron transport system I. photoreduction and photo-oxidation of cytochrome b559 by photosystem II in spinach chloroplasts

Light-induced redox-reactions of cytochrome b₅₅₉ in spinachchloroplasts were investigated. Illumination of chloroplastsinduced photoreduction of cytochrorne b₅₅₉ Red light (650 nm)was more effective than far-red light (725 nm), indicating thatthe photoreduction is a photosystem II-mediated reaction. Onaddition of DCMU, the photoreduction was eliminated and a photooxidationof cytochrome b₅₅₉ was observed. The rate of this photooxidationwas faster with photosystem II light than with photo-systemI light. On addition of Mn⁺⁺ the photooxidation was partly suppressed;far-red light became as effective as red light in inducing photooxidationof cytochrome b₅₉₉, in the presence of DCMU and Mn⁺⁺. Ascorbate completely suppressed photooxidation of cytochromeb⁵⁵⁹ In the presence of ascorbate, however, photooxidation wasobserved in the presence of inhibitors or after inhibitory treatmentsof chloroplasts which affected the oxidizing side of systemII. These inhibitors and inhibitory treatments, but not DCMU,decreased the redoxpotential of cytochrome b₅₅₉. Reactivationof Hill reaction in Tris-washed chloroplasts by indophenol-ascorbatetreatment was not accompanied by an abolishment of photooxidationof cytochrome b₅₅₉. A possible mechanism is proposed to account for these reactionsof cytochrome b₅₅₉ in the photosynthetic electron transportin chloroplasts. (Received April 4, 1972; )     

Role of Ca2+ on Triggering Tonoplast Action Potential in Intact Nitella flexilis

Sensitive aequorin was microinjected into the cytoplasm of Nitellaflexilis to study the role of Ca²⁺ in the generation of thetonoplast action potential. The temporal relation between theincrease in cytoplasmic Ca²⁺ and the tonoplast action potentialsuggested that tonoplast action potential is triggered by anincrease in cytoplasmic Ca²⁺. This is also supported by thefact that Mn²⁺, extracellularly applied, inhibited both theincrease in cytoplasmic Ca²⁺ and the generation of the tonoplastaction potential. (Received April 2, 1997; Accepted June 7, 1997)     

Flash reactivation of Tris-inactivated chloroplasts

The light conditions required for reactivation of the oxygen-evolvingsystem in Tris-treated chloroplasts were studied by means ofrepetitive flashes. Inactive Tristreated chloroplasts were washedwith reduced 2,6-dichlorophenolindophenol, suspended in a reactivationmedium containing Mn²⁺⁺ and Ca²⁺⁺ ions, then illuminated withflashes. Flashes at dark intervals of 2 sec were most effectivefor reactivation, while those at shorter or longer intervalswere less effective. It was deduced that more than two sequentiallight reactions with dark reactions in between were involvedin the reactivation. (Received December 28, 1977; )     

Divalent metal cation requirements of phosphofructokinase-2 from E. coli. Evidence for a high affinity binding site for Mn

The reaction catalyzed by E. coli Pfk-2 presents a dual-cation requirement. In addition to that chelated by the nucleotide substrate, an activating cation is required to obtain full activity of the enzyme. Only Mn²⁺ and Mg²⁺ can fulfill this role binding to the same activating site but the affinity for Mn²⁺ is 13-fold higher compared to that of Mg²⁺. The role of the E190 residue, present in the highly conserved motif NXXE involved in Mg²⁺ binding, is also evaluated in this behavior. The E190Q mutation drastically diminishes the kinetic affinity of this site for both cations. However, binding studies of free Mn²⁺ and metal–Mant-ATP complex through EPR and FRET experiments between the ATP analog and Trp88, demonstrated that Mn²⁺ as well as the metal–nucleotide complex bind with the same affinity to the wild type and E190Q mutant Pfk-2. These results suggest that this residue exert its role mainly kinetically, probably stabilizing the transition state and that the geometry of metal binding to E190 residue may be crucial to determine the catalytic competence.     

Linolenic acid binding by chloroplasts

The binding of linolenic acid with chloroplasts was investigatedwith ¹⁴C-labelled linolenic acid. The effect of the fatty acidon the activity of the electron transport system was also studied. The amount of linolenic acid bound to chloroplasts increasedwith increasing concentration of the fatty acid added in a mannersuggesting a cooperative mode of binding. At the highest concentrationof linolenic acid added (100 µM), the molar ratio of boundfatty acid to chlorophyll was four. The bound fatty acid to chlorophyll ratios were inversely proportionalto the amounts of chloroplasts added. The bound fatty acid wasreleased by addition of bovine serum albumin or by washing ofthe chloroplasts. The mode of release during repeated washingindicates that binding of linolenic acid to chloroplast membraneoccurred through partition of the fatty acid between the membraneand the aqueous medium. Time courses and temperature dependency of the development oflinolenic acid-induced inhibition of the Hill reaction weremarkedly different from those of the fatty acid binding. Theinhibition was at least partially reversible. The results indicatethat inactivation of electron transport is due to disorganizationof the functional integrity of the membrane caused by penetrationof the fatty acid molecules into the hydrophobic region of membrane. ¹ Present address: Biological Laboratory, Nippon Medical School,Kosugi, Kawasaki, Japan. (Received December 16, 1976; )     

The Divalent Cation Pump and its Role in the Regulation of Malic Enzyme Activity in Purified Mitochondria from Potato Tuber

The control of the activity of the matrix-located malic enzyme(EC 1.1.1.39 [EC] ) by Mn²⁺ was investigated in Percoll-purified mitochondriafrom potato (Solarium tuberosum) tuber. Malic enzyme activitywas tightly controlled by the amount of Mn²⁺ available in thematrix space and could be stimulated by the addition of exogenousMn²⁺. A net uptake of Mn²⁺ into the matrix space of energizedmitochondria was measured. The uptake of Mn²⁺ was mediated bythe active cation pump present in the mitochondria. The activityof this cation pump was shown to be dependent on the membranepotential sustained by the activity of the respiratory chain.The uptake of Mn²⁺ was totally abolished in the presence ofan uncoupler and strongly depressed in the presence of rutheniumred, a specific inhibitor of the Ca²⁺-pump which is presentin animal mitochondria. Thus, the effect of Mn²⁺ on matrix-locatedMn²⁺-dependent malic enzyme was strongly influenced by the presenceof an uncoupler or of ruthenium red. In addition, this effectwas reduced in the presence of Ca²⁺. The possible physiologicalsignificance of the presence of this cation pump is discussedin relation to the presence of a matrix-located, NAD⁺-dependentmalic enzyme in plant mitochondria. (Received November 21, 1988; Accepted March 6, 1989)     

Serotonin binding protein:enhancement of binding by Fe2+ and inhibition of binding by drugs.

The binding of serotonin to a soluble, high affinity binding protein, present in synaptosomes and associated with serotonergic tracts, has now been studied for the effects of metallic ions and various drugs. At optimal concentration (10^-4 M) of Fe²⁺ the enhancement of binding was close to 20-fold. A much smaller effect was noted with Cu²⁺. With other ions (Fe³⁺, Mn²⁺, Co²⁺, Ni²⁺, Cr³⁺, Mg²⁺, Ca²⁺) little or no effect was seen. For the effect with Fe²⁺. preincubation was required (10 min, 25°C) and concentrations higher than 10^-4M were inhibitory. Studies based on equilibrium dialysis show that the effect of Fe²⁺ was on the affinity of the binding of serotonin to the protein, rather than on the binding capacity. In polydcrylamide gels at pH 8.6 the migratory properties of thc serotonin-protein complex formed in the presence of Fe²⁺ differ from those of the complex formed without Fe²⁺. Nucleotides (ATP, GTP, ADP, AMP) inhibited thc binding. The effects of several classes of drugs (inhibitors of biogenic amine storage and uptake, psychotomimetics, MAO) inhibitors and drugs binding to contractile proteins) were also studied. The only effective inhibitors of serotonin binding were reserpine, vinblastine and CZ-74, which caused 50% inhibition at 2 × 10^-6 M, 7.5 × 10^-6 M and 0.2 × 10^-6M respectively.