Field-dispersion profiles of the proton spin-lattice relaxation rate in chloroplast suspensions. Effect of manganese extraction by EDTA,Tris, and hydroxylamine

Proton spin-lattice relaxation rates (R₁) have been measured in a variety of dark-adapted chloroplast suspensions over a range of field strengths between 1 and 15 kG (4–5 MHz). When the effects of EDTA or Tris washing on chloroplast relaxivities are compared, the pool of Mn associated with oxygen evolution is seen not to contribute significantly to relaxivity. Instead, nearly all of the observed relaxivity, which is characterized by a paramagnetic maximum near 20.7 MHz in the field dispersion profile of R₁, appears to arise from contaminating non-functional Mn(II) that can be removed by EDTA during the isolation procedure. These observations, which contradict previous reports ascribing chloroplast relaxivity to the water-oxidizing system, require a reevaluation of proposed models, derived from NMR studies, of the state of Mn in the water-splitting reaction.Chloroplasts from which loosely bound non-functional Mn has been removed by EDTA washing do show an enhancement of relaxivity when exposed to NH₂OH at concentrations known to inactivate water oxidation. This NH₂OH-induced relaxivity is comprised of Mn(II) in two distinct paramagnetic sites. One site is chelatable by EDTA, whereas the other site is not. This finding suggests that some Mn(II) tightly bound to thylakoid membranes can contribute to relaxivity after inactivation of the oxygen-evolving reaction.     

Effect of manganese on the nuclear magnetic relaxivity of water protons in chloroplast suspensions

Field dispersion profiles of the proton spin-lattice relaxation rate, T1^?1, in chloroplast suspensions show a local maximum near 20 MHz, probably due to bound Mn(II); EDTA extraction eliminates, and MnCl₂ addition restores, the paramagnetic relaxivity. Since neither treatment affects water oxidation, the Mn(II) site monitored appears to lie outside the water-splitting enzyme. Intense illumination almost totally suppresses the paramagnetic relaxivity through an electron-transport-dependent mechanism. Previous reports that chloroplast nuclear magnetic relaxivity varies cyclically in flash experiments require reevaluation in terms of the probable role of Mn(II) that is nonfunctional in water oxidation.     

Effects of la on surface charges, dielectrophoresis, and electrofusion of barley protoplasts

When dielectrophoresis and electrofusion of barley (Hordeum vulgare var Moor) leaf protoplasts were assayed in the presence of 0.1 to 1 millimolar lanthanum ion (La³⁺) in the basal medium (0.7 molar mannitol, 1 millimolar piperazine-N, N-bis[2-ethanesulfonic acid]-Na [pH 6.7], 0.1 millimolar CaCl₂), dielectrophoresis and induction of electrofusion were strongly inhibited. The latter remained inhibited and the former recovered by about 60% after washing the La³⁺ -treated protoplasts without EDTA. These inhibitions were almost completely abolished by washing the La³⁺ -treated protoplasts with 1 millimolar EDTA. Inductively coupled plasma atomic emission spectroscopic analysis revealed that protoplasts retained a considerable amount of La³⁺ after washing without EDTA and released most of the bound La³⁺ by washing with 1 millimolar EDTA. This tightly bound La³⁺ seemed responsible for the inhibition of electrofusion and dielectrophoresis that was observed in the La³⁺ -treated protoplasts after washing. ζ-potentials of protoplasts were -39.0±3.2 millivolts, -16.7 ± 2.6 millivolts, and virtually zero in media containing 0, 0.1, and 0.3 millimolar La³⁺ (I = 7.2 millimolar), respectively, and had a positive value (+ 14.2 ± 2.2 millivolts) in the presence of 1 millimolar La³⁺. These effects of La³⁺ on ζ-potentials were easily abolished by washing without EDTA. This indicates that charged species located at the surface of plasma membrane of protoplasts cannot account for the sites at which La³⁺ exerts its inhibition of dielectrophoresis and electrofusion. In contrast, the promotion of spherical fusion and the reduction of broken fusion products observed in the presence of La³⁺ were almost completely abolished by washing without EDTA. Our results also indicate that the initial induction and development of electrofusion can be studied independently.     

Water proton relaxation as a monitor of membrane-bound manganese in spinach chloroplasts.

First measurements of proton relaxation on chloroplast membranes are presented here. Experiments show that the water proton spin-lattice relaxation rate in chloroplast thylakoid membrane suspensions can be used to monitor membrane-bound manganese. The relaxation effect is reduced to 0.4 of its original value upon manganese extraction by washing with either alkaline Tris buffer or NH2OH/EDTA solution. Large increases in the proton relaxation rate are measured in the presence of reductants such as tetraphenylboron and NH2OH; oxidants such as potassium ferricyanide or 2,6-dichlorophenolindophenol lead to an decrease in this rate. These results suggest that manganese exists as a mixture of oxidation states in dark-adapted chloroplasts.     

The photosynthetic water oxidase: its proton pumping activity is short-circuited within the protein by DCCD

The photosynthetic water oxidase is composed of ˜15 polypeptides which are grouped around two functional parts: photosystem II and the catalytic manganese centre. Photochemically driven vectorial electron transfer between the manganese centre and bound plastoquinone causes deprotonation–protonation reactions at opposite sides of the thylakoid membrane. Thereby the water oxidase acts as a proton pump. Incubation of stacked thylakoids with N,N'-dicyclohexylcarbodiimide (DCCD) short-circuited its proton pumping activity. Under flashing light, the extent of both proton release into the lumen by water oxidation and of proton uptake from the medium by reduced quinone was diminished. Instead there was a rapid electrogenic backreaction with a strong H/D-isotope effect. Apparently protons which were produced by water oxidation were channelled across the transmembrane protein to the bound quinone. A more rapid protonation of the reduced quinone was evident from a shortening of the time lag for the reduction of photosystem I. These effects were paralleled by the preferential labelling with [¹⁴C]DCCD in stacked thylakoids of two polypeptides with 20 and 24 kd apparent molecular mass. These may be capping the oxidizing and the reducing terminus of the water oxidase to control proton extrusion and proton uptake respectively.     

A method for studying the cellular location of lead in lichens

Abstract: A number of possible displacing agents have been tested for their ability to release extracellular bound lead from Cladonia portentosa. Metal cations (Ni and Cu), thiol-rich reagents (glutathione, ammonium pyrrolidinedithiocarbamate) and some chelating agents (EGTA, EDTA at pH 9·5 or pH 2·7) were ineffective or caused membrane damage. Disodium EDTA at pH 4·5 was found to be an effective displacing agent, although the Na caused some release of intracellular K without membrane damage. This displacement procedure showed that no Pb supplied in the laboratory entered the protoplast in 2 h. The effect of Ca, Cu, Pb, and Sr on membrane integrity was investigated.     

Synthesis and in vitro and in vivo evaluation of manganese(III) porphyrin–dextran as a novel MRI contrast agent

5-(4-Aminophenyl)-10,15,20-tris(4-sulfonatophenyl) manganese(III) porphyrin conjugated with dextran was synthesized. Its potential of being used as a tumor-targeting magnetic resonance imaging contrast agent was evaluated in vitro and in vivo. The results demonstrated that the compound has a longitudinal relaxivity (R₁) higher than Gd-DTPA, low cytotoxicity and binding specificity to tumor cell membrane.     

Chloroplast manganese and superoxide.

Particles prepared from spinach chloroplast membranes with Triton X-100 inhibited the superoxide-mediated reduction of nitro-blue tetrazolium by riboflavin. This superoxide dismutase-like activity was of two kinds, one inactivated by heating and inhibited by H₂O₂ and the other insensitive to both of these treatments; both activities were destroyed by washing with concentrated Tris buffer or with EDTA. Attempts at reconstitution with transition metal ions suggested that two different forms of bound manganese may be responsible and it is proposed that the inhibition by H₂O₂ is indicative of three different oxidation states of particle-bound manganese. The possibility that the photosynthetic water-splitting system and superoxide dismutase have evolved from a single precursor is discussed.     

Tightly bound nucleotides of the energy-transducing ATPase,and their role in oxidative phosphorylation. I. The Paracoccus denitrificans system

1. The coupling ATPase of Paracoccus denitrificans can be removed from the membrane by washing coupled membrane fragments at low salt concentrations.2. This ATPase resembles coupling ATPases of mitochondria, chloroplasts and other bacteria. It is a negatively charged protein of molecular weight about 300 000. An inhibitor protein is bound tightly to the ATPase in vivo, and can be destroyed by trypsin treatment.3. ATP and ADP are found tightly bound to the coupling ATPase of P. denitrificans, both in its membrane-bound and isolated state. The ATP/ADP ratio on the enzyme is greater than one.4. Under de-energised conditions, the bound nucleotides are not available to the suspending medium. When the membrane is energised however, the bound nucleotides can exchange with added nucleotides and incorporate ³²P_i. ³²P_i is incorporated into the β and γ positions of the bound nucleotides, but β-labelling probably does not occur on the coupling ATPase.5. Uncouplers inhibit the exchange of the free nucleotides or ³²P_i into the bound nucleotides, while venturicidin (an energy transfer inhibitor) and aurovertin stimulate the exchange.6. The response of the bound nucleotides to energisation is consistent with their being involved directly in the mechanism of oxidative phosphorylation.     

Water proton relaxation as a monitor of membrane-bound manganese in spinach chloroplasts

First measurements of proton relaxation on chloroplast membranes are presented here. Experiments show that the water proton spin-lattice relaxation rate in chloroplast thylakoid membrane suspensions can be used to monitor membrane-bound manganese. The relaxation effect is reduced to 0.4 of its original value upon manganese extraction by washing with either alkaline Tris buffer or NH₂OH/EDTA solution. Large increases in the proton relaxation rate are measured in the presence of reductants such as tetraphenylboron and NH₂OH; oxidants such as potassium ferricyanide or 2,6-dichlorophenolindo-phenol lead to a decrease in this rate. These results suggest that maganese exists as a mixture of oxidation states in dark-adapted chloroplasts.     

Accumulation and precipitation of Mn2+ by the cells of Oscillatoria terebriformis

The cyanobacterium Oscillatoria terebriformis was shown to exhibit resistance to high manganese concentrations, remaining viable at 2.5 mM MnCl₂ in the medium. Cyanobacterial cells were capable of considerable manganese consumption from the medium. The dynamics of Mn sorption by the cells were the same in all experimental variants, independent of the manganese concentration. Manganese concentration in the biomass peaked after 2–3 days and depended on Mn²⁺ concentration in the medium and on the amount of biomass introduced. In the case of O. terebriformis, manganese removed from the medium may be subdivided into Mn absorbed by the cell, Mn bound to the cell wall, Mn absorbed by the glycocalix, and chemically precipitated Mn. Of the total 21.25 ± 1.0 mg of consumed manganese, biological absorption and chemical precipitation were responsible for 11.78 ± 0.98 and 9.2 ± 0.8 mg, respectively. In the presence of cyanobacteria, Mn removal from the medium was 2.28 times higher than in the control. This process depended considerably on Mn sorption by exopolysaccharides. At 1.3 mM Mn²⁺, a lamellar mat was formed with interlayers of manganese carbonate.     

The nature and location of Acholeplasma laidlawii membrane proteins investigated by two-dimensional gel electrophoresis

The high resolution, two-dimensional electrophoresis system for the separation of proteins described by O'Farrell, (O'Farrell, P.H. (1975) J. Biol. Chem. 250, 4007–4021) has been modified for the separation of Acholeplasma laidlawii proteins.Reproducible protein patterns have been obtained from A. laidlawii cell, membrane and soluble protein preparations. The isoelectric focusing of membrane proteins was greatly improved by removing the bulk of the membrane lipid before solubilizing the protein.A. laidlawii peripheral membrane proteins were removed from the membrane by low ionic strength washing and by treatment with EDTA. The effect of an exhaustive EDTA treatment and a rapid, warm EDTA treatment were compared. By comparing the protein patterns obtained in these ways it was possible to distinguish two separate groups of peripheral membrane proteins and one integral membrane protein group. The peripheral membrane proteins which were removed from the membrane at low ionic strength (group I) were also insoluble in Triton X-100, whereas additional peripheral membrane proteins extractable by subsequent EDTA treatment (group II) were soluble in Triton X-100.Exterior-facing membrane proteins were distinguished from the interiorfacing ones by lactoperoxidase-catalyzed iodination of intact cells and membranes. Group I peripheral membrane proteins faced the cell interior whereas group II proteins faced the cell exterior. We counted approximately 320 individual whole cell proteins. Of these, about 140 were membrane associated and a maximum of 40 proteins were iodinated after iodinationg intact cells.A. laidlawii was also grown in the presence of NaH₂³²PO₄ and whole cell proteins were separated by two-dimensional gel electrophoresis. One membrane protein and two soluble proteins were labelled.     

Manganese(III) porphyrins complexed with P22 virus-like particles as T 1-enhanced contrast agents for magnetic resonance imaging

Virus-like particles are powerful platforms for the development of functional hybrid materials. Here, we have grown a cross-linked polymer (cross-linked aminoethyl methacrylate) within the confines of the bacteriophage P22 capsid (P22–xAEMA) and functionalized the polymer with various loadings of paramagnetic manganese(III) protoporphyrin IX (MnPP) complexes for evaluation as a macromolecular magnetic resonance imaging contrast agent. The resulting construct (P22–xAEMA–MnPP) has r _1,particle = 7,098 mM^?1 s^?1 at 298 K and 2.1 T (90 MHz) for a loading of 3,646 MnPP molecules per capsid. The Solomon–Bloembergen–Morgan theory for paramagnetic relaxivity predicts conjugating MnPP to P22, a supramolecular structure, would result in an enhancement in ionic relaxivity; however, all loadings experienced low ionic relaxivities, r _1,ionic, ranging from 1.45 to 3.66 mM^?1 s^?1, similar to the ionic relaxivity of free MnPP. We hypothesize that intermolecular interactions between neighboring MnPP molecules block access of water to the metal site, resulting in low r _1,ionic relaxivities. We investigated the effect of MnPP interactions on relaxivity further by either blocking or exposing water binding sites on MnPP. On the basis of these results, future design strategies for enhanced r _1,ionic relaxivity are suggested. The measured r _2,ionic relaxivities demonstrated an inverse relationship between loading and relaxivity. This results in a loading-dependent r ₂/r ₁ behavior of these materials indicating synthetic control over the relaxivity properties, making them interesting alternatives to current magnetic resonance imaging contrast agents.     

Effects of p-Aminosalicylic acid on the neurotoxicity of manganese on the dopaminergic innervation of the cilia of the lateral cells of the gill of the bivalve mollusc, Crassostrea virginica

The lateral cilia of the gill of Crassostrea virginica are controlled by a dopaminergic–serotonergic innervation. Dopamine is the neurotransmitter causing cilio-inhibition. High levels of manganese are neurotoxic to people, causing Manganism, a Parkinson-like disease. Clinical interventions for Manganism have not been very successful. Recently, p-Aminosalicylic acid (PAS) was reported as an effective treatment of severe Manganism in humans; however, its mechanism of action is unknown. Previously, we reported that manganese treatments caused disruption of the dopaminergic innervation of gill of C. virginica. Here we compared the effects of manganese on gill innervation in the presence of PAS, EDTA or Acetylsalicylic acid (ASA), and examined whether co-treating animals with PAS could block the deleterious effects of manganese on the oyster's dopaminergic innervation of the gill. Beating rates of the lateral cilia of the gill were measured by stroboscopic microscopy. Pre-treating gill preparations with PAS or EDTA blocked the neurotoxic effects of manganese, while ASA did not. In other experiments, animals exposed to three day treatments with manganese produced a dose dependent impairment of the dopaminergic, cilio-inhibitory system, which was decreased by co-treatment with PAS. The study shows that PAS protects the animal against neurotoxic effects of manganese and the mechanism of action of PAS in alleviating Manganism is more likely related to its chelating abilities than its anti-inflammatory actions.     

A spectral shift in cytochrome a induced by calcium ions

Ca²⁺ induces a red shift in the absorption spectrum of ferrocytochrome a when added to uncoupled mitochondria, sub-mitochondrial particles or isolated cytochrome aa₃. The shift is identical within experimental error to the previously reported energy-linked shift in intact mitochondria (Wikström, M. K. F. (1972), Biochim. Biophys. Acta 283, 385–390). One mol of calcium produces the shift in one mol of cytochrome a, the K_D being approx. 20–30 μM. The calcium-induced shift is readily reversed by chelating agents such as EDTA, ethyleneglycol-bis-(μ-aminoethyl ether)N,N′-tetraacetic acid (EGTA) and ATP and is insensitive to uncoupling agents and inhibitors of calcium transport (La³⁺ and ruthenium red). It is shown that the binding site for calcium that is responsible for the spectral shift is located on the outside of the permeability barrier of the mitochondrial cristae membrane.It is proposed that calcium simulates the energy-linked shift in cytochrome a by binding to a site of cytochrome aa₃ that is occupied by protons in energized mitochondria and that is located at the external surface of the mitochondrial membrane.     

The Discrimination between Magnesium and Manganese by Serum Proteins

Magnesium and manganese have proved physically and functionally interchangeable in many isolated biological systems investigated in vitro. This lack of discrimination contrasts sharply with the high biological specificity exhibited by intact mammals under a large variety of conditions. The dichotomy between intact animals and their isolated systems might be due at least partially to presence vs. absence of an intact circulation. Hence the capability of mammalian plasma to discriminate between the alkaline earth and the transition metal was investigated by means of equilibrium dialysis, exchange, ultrafiltration, ultracentrifugation, and zone electrophoresis. The states of the respective elements are thus contrasted as follows: (a) Magnesium is partially bound, manganese totally. (b) Magnesium is nonselectively bound by serum proteins, manganese selectively by a β₁-globulin. (c) Under conditions approaching physiological, the two metals do not interchange. This is interpreted as indicating that the plasma proteins contribute to biological specificity by discriminating between a trace metal and a macronutrient.     

Inhibition of Nitrate Transport by Anti-Nitrate Reductase IgG Fragments and the Identification of Plasma Membrane Associated Nitrate Reductase in Roots of Barley Seedlings

Membrane associated nitrate reductase (NR) was detected in plasma membrane (PM) fractions isolated by aqueous two-phase partitioning from barley (Hordeum vulgare L. var CM 72) roots. The PM associated NR was not removed by washing vesicles with 500 millimolar NaCl and 1 millimolar EDTA and represented up to 4% of the total root NR activity. PM associated NR was stimulated up to 20-fold by Triton X-100 whereas soluble NR was only increased 1.7-fold. The latency was a function of the solubilization of NR from the membrane. NR, solubilized from the PM fraction by Triton X-100 was inactivated by antiserum to Chlorella sorokiniana NR. Anti-NR immunoglobulin G fragments purified from the anti-NR serum inhibited NO₃⁻ uptake by more than 90% but had no effect on NO₂⁻ uptake. The inhibitory effect was only partially reversible; uptake recovered to 50% of the control after thorough rinsing of roots. Preimmune serum immunoglobulin G fragments inhibited NO₃⁻ uptake 36% but the effect was completely reversible by rinsing. Intact NR antiserum had no effect on NO₃⁻ uptake. The results present the possibility that NO₃⁻ uptake and NO₃⁻ reduction in the PM of barley roots may be related.     

The effect of Zn2+ on the inhibition of Fru-P2ase by AMP.

In the absence of chelating agents, the sensitivity of rabbit liver fructose 1,6-bisphosphatase to inhibition by AMP is increased by approximately 10-fold, apparently related to the presence of bound Zn²⁺. At pH 7.5, 10 μM AMP causes virtually complete inhibition and nearly full activity is restored by the addition of chelating agents such as EDTA or histidine. These properties provide a mechanism for the induction of Fru-P₂ase activity under gluconeogenic conditions.     

Testosterone metabolism by homogenates of human prostates with benign hyperplasia: Effects of zinc,cadmium and other bivalent cations

The effects of various quantities of Ba, Be, Ca, Cd, Co, Cu, Mg, Mn, Sr, Zn and EDTA on the formation of 5α-reduced metabolites of testosterone (T) substrate and of 3α-/3β -reduced metabolites of 5α-dihydrotestosterone substrates by homogenates of 6 human hyperplastic prostate glands were studied in incubations at pH 7.4 with NADPH-generating system. Effects of these cations and EDTA on the V_M and K_M of the 5α-reductase and 3α-/3β-hydroxysteroid dehydrogenases (-HSD) were also measured. Quantities of 5α-reduced T metabolites were significantly increased by Cd, Cu and Zn supplementations. These increments were shown to result from significant augmentations of the V_M but no change in K_M of the NADPH-dependent 5α -reductase. Quantities of 3α -reduced DHT metabolites were significantly decreased by Cd and Cu supplementations and resulted from an increase of the K_M of the NADPH-dependent 3α-HSD by Cd and both an increase of K_M and a decrease of V_M by Cu. Quantities of β-reduced DHT metabolites were significantly decreased by Cd and Cu supplementations. Increase of the K_M of the NADPH-dependent 3β-HSD by Cd was found significant while Cu both increased the Am and decreased the V_M of the enzyme. EDTA-related changes in 5α-reductase activity were shown to result from the EDTA-induced decrease of the pH of the medium. No effect of EDTA was observed on the activities of both 3α/3β-HSD.     

Effect of heavy metal pollution on mineral absorption in sunflower (Helianthus annuus L.) hybrids

The present study was conducted in a potted experiment to examine the effects of chromium pollution on absorption of mineral nutrients and some morpho-physiological attributes of two sunflower (Helianthus annuus L.) hybrids (FH-331 and FH-259) in the presence and absence of ethylene diamine tetra acetic acid (EDTA) used as a chelating agent. Four concentrations of chromium (Cr³⁺) i.e., 0, 20, 30 and 40 mg kg^?1 with and without 0.3 g kg^?1, EDTA as chelating agent were applied to 25-day-old sunflower plants. A gradually decreasing trend in absorption of all minerals and other parameters studied were observed. Different treatments of Cr³⁺ as well as Cr³⁺ and EDTA significantly reduced root and shoot fresh weight; however, root, shoot and achene Cr³⁺ contents of two sunflowers hybrids under higher chromium and EDTA stress varied significantly whereas movement of Cr³⁺ contents to leaves was non-significant. Absorption of Na⁺, K⁺, N₂ and P through roots and shoots significantly reduced with increasing concentration of Cr³⁺ treatments. In fact addition of EDTA to the medium further enhanced the toxicity of chromium.