The physiological versus the spectrophotometric status of phytochrome in corn coleoptiles

The influence of red light in altering the phototropic sensitivity of corn coleoptiles (Zea mays L., cultivar Burpee Barbecue Hybrid) is compared with the spectrophotometric status of the phytochrome they contain. The distribution of measurable phytochrome corresponds roughly with the distribution of sensitivity to red light for physiological change. Both phytochrome concentration and red light sensitivity are maximal in the coleoptile tips. Red light pretreatments which reduce total phytochrome by about 50%, however, do not alter subsequent red light sensitivity of the phototropic system. Dosages of red light sufficient to saturate the physiological system are two orders of magnitude too small to induce measurable phytochrome transformation. The log-dosage-response curves for physiological change and for phytochrome transformation do not have the same slopes. The time course for appearance, mainconcentration of the far-red-absorbing form of phytochrome over a broad range of tenance, and decay of the physiological response is independent of the measurable concentrations. The following hypothesis is proposed: the phytochrome mediating the alteration in phototropic sensitivity is only a small proportion of the total present. This small active fraction is physically and kinetically independent of the bulk measurable, and is packaged in some manner which facilitates its transformation in both directions.     

Molecular dissection of interactions between components of the alternative pathway of complement and decay accelerating factor (CD55)

The complement regulatory protein decay accelerating factor (DAF; CD55), inhibits the alternative complement pathway by accelerating decay of the convertase enzymes formed by C3b and factor B. We show, using surface plasmon resonance, that in the absence of Mg(2+), DAF binds C3b, factor B, and the Bb subunit with low affinity (K(D), 14 +/- 0.1, 44 +/- 10, and 20 +/- 7 microm, respectively). In the presence of Mg(2+), DAF bound Bb or the von Willebrand factor type A subunit of Bb with higher affinities (K(D), 1.3 +/- 0.5 and 2.2 +/- 0.1 microm, respectively). Interaction with the proenzyme C3bB was investigated by flowing factor B across a C3b-coated surface in the absence of factor D. The dissociation rate was dependent on the time of incubation, suggesting that a time-dependent conformational transition stabilized the C3b-factor B interaction. Activation by factor D (forming C3bBb) increased the complex half-life; however, the enzyme became susceptible to rapid decay by DAF, unlike the proenzyme, which was unaffected. A convertase assembled with cobra venom factor and Bb was decayed by DAF, albeit far less efficiently than C3bBb. DAF did not bind cobra venom factor, implying that Bb decay is accelerated, at least in part, through DAF binding of this subunit. It is likely that DAF binds the complex with higher affinity/avidity, promoting a conformational change in either or both subunits accelerating decay. Such analysis of component and regulator interactions will inform our understanding of inhibitory mechanisms and the ways in which regulatory proteins cooperate to control the complement cascade.     

Multivalent metal-induced iron acquisition from transferrin and lactoferrin by myeloid cells

We previously described a unique, high-capacity, ATP-independent mechanism through which myeloid cells acquire Fe from low-m.w. chelates. The rate of this Fe acquisition is markedly increased by cellular exposure to multivalent metal cations. Because most Fe in vivo is bound to transferrin or lactoferrin, we examined whether this mechanism also contributes to myeloid cell acquisition of Fe from transferrin and/or lactoferrin. Using HL-60 cells as a model system, we show cellular acquisition of (59)Fe from both lactoferrin and transferrin that was unaffected by conditions that depleted the cells of ATP or disrupted their cytoskeleton. Fe acquisition was dramatically increased by cell exposure to various metals including Ga(3+), Gd(3+), Al(3+), Fe(3+), La(3+), Zr(4+), Sn(4+), Cu(2+), and Zn(2+) by a process that was reversible. Exposure to these same metals also increased binding of both transferrin and lactoferrin to the cell surface by a process that does not appear to involve the well-described plasma membrane receptor for transferrin. Approximately 60% of the Fe acquired by the cells from transferrin and lactoferrin remained cell associated 18 h later. HL-60 cells possess a high-capacity multivalent metal-inducible mechanism for Fe acquisition from transferrin and lactoferrin that bears many similarities to the process previously described that allows these and other cell types to acquire Fe from low-m.w. Fe chelates. The biologic importance of this mechanism may relate to its high Fe acquisition capacity and the speed with which it is able to rapidly adapt to the level of extracellular Fe.     

Membrane potential-controlled inhibition of cytochrome c oxidase by zinc

Like many voltage-sensitive ion pumps, cytochrome c oxidase is inhibited by zinc. Binding of zinc to the outside surface of Rhodobacter sphaeroides cytochrome c oxidase inhibits the enzyme with a K(I) of < or = 5 microm when the enzyme is reconstituted into phospholipid vesicles in the presence of a membrane potential. In the absence of a membrane potential and a pH gradient, millimolar concentrations of zinc are required to inhibit. This differential inhibition causes a dramatic increase in the respiratory control ratio from 6 to 40 for wild-type oxidase. The external zinc inhibition is removed by EDTA and is not competitive with cytochrome c binding but is competitive with protons. Only Cd(2+) of the many metals tested (Mg(2+), Mn(2+), Ca(2+), Ba(2+), Li(2+), Cs(2+), Hg(2+), Ni(2+), Co(2+), Cu(2+) Tb(3+), Tm(3+)) showed inhibitory effects similar to Zn(2+). Proton pumping is slower and less efficient with zinc. The results suggest that zinc inhibits proton movement through a proton exit path, which can allow proton back-leak at high membrane potentials. The physiological and mechanistic significance of proton movement in the exit pathway and its blockage by zinc is discussed in terms of regulation of the efficiency of energy transduction.     

N-terminal domain of Avena phytochrome: interactions with sodium dodecyl sulfate micelles and N-terminal chain truncated phytochrome.

Phytochrome is the ubiquitous red light photoreceptor present in plants. Properties of the 6-kDa end terminal region of phytochrome A (PHYA from etiolated Avena) have been investigated by the use of synthetic polypeptide fragments corresponding to that region. This region of the phytochrome A protein has been viewed as a possible functional site due to the large differences in the sequence's conformation and exposure between the Pr (red light-absorbing form) and Pfr (far-red light-absorbing, gene-regulating form) species of phytochrome A. Hydrophobic moment calculations reveal amphiphilic helical potential in this section of the protein, consistent with the folding of the N-terminal region onto a hydrophobic chromophore/chromophore pocket. A large N-terminal synthetic peptide also demonstrated helical folding in the presence of SDS micelles. This experimental evidence indicates that the N-terminal alpha-helical folding upon conversion of the regulatorily inactive Pr to the active Pfr form of phytochrome A is likely driven at least in part by amphiphilic helix stabilization. Further, the large synthetic peptide was spectrally demonstrated to interact with phytochrome A lacking the N-terminal region. The formation of this nativelike complex may provide us with a tool for both biophysical and physiological studies on the mechanism of phytochrome A signal transduction.     

Viscosity Dependence of Primary Photoprocesses of 124 Kilodalton Phytochrome

To characterize the nature of primary photoprocesses of phytochrome which serves as the red-far red reversible photoreceptor for photomorphogenesis in plants, viscosity dependence of the fluorescence lifetimes of phytochrome isolated from etiolated oat seedling (Avena sativa L.) has been investigated. The fluorescence decay of phytochrome exhibited approximately two components, one with lifetime in the range of 50-70 ps and another with 1.1-1.2 ns in phosphate buffer with or without 40-67% glycerol. However, relative amplitudes of these decay components were found to be strongly viscosity dependent. Thus, the longer decay component increased from 2-5% in phosphate buffer to ~20% in 67% glycerol-phosphate buffer. These results have been interpreted in terms of primary reaction from the excited singlet state of phytochrome, yielding a photoreversible intermediate whose rate of formation and decay were apparently viscosity-dependent. Further, the viscosity dependence is consistent with the primary reaction involving conformational changes of the chromophore/its apoprotein environment.     

TRPM7 provides an ion channel mechanism for cellular entry of trace metal ions

Trace metal ions such as Zn(2+), Fe(2+), Cu(2+), Mn(2+), and Co(2+) are required cofactors for many essential cellular enzymes, yet little is known about the mechanisms through which they enter into cells. We have shown previously that the widely expressed ion channel TRPM7 (LTRPC7, ChaK1, TRP-PLIK) functions as a Ca(2+)- and Mg(2+)-permeable cation channel, whose activity is regulated by intracellular Mg(2+) and Mg(2+).ATP and have designated native TRPM7-mediated currents as magnesium-nucleotide-regulated metal ion currents (MagNuM). Here we report that heterologously overexpressed TRPM7 in HEK-293 cells conducts a range of essential and toxic divalent metal ions with strong preference for Zn(2+) and Ni(2+), which both permeate TRPM7 up to four times better than Ca(2+). Similarly, native MagNuM currents are also able to support Zn(2+) entry. Furthermore, TRPM7 allows other essential metals such as Mn(2+) and Co(2+) to permeate, and permits significant entry of nonphysiologic or toxic metals such as Cd(2+), Ba(2+), and Sr(2+). Equimolar replacement studies substituting 10 mM Ca(2+) with the respective divalent ions reveal a unique permeation profile for TRPM7 with a permeability sequence of Zn(2+) approximately Ni(2+) > Ba(2+) > Co(2+) > Mg(2+) >/= Mn(2+) >/= Sr(2+) >/= Cd(2+) >/= Ca(2+), while trivalent ions such as La(3+) and Gd(3+) are not measurably permeable. With the exception of Mg(2+), which exerts strong negative feedback from the intracellular side of the pore, this sequence is faithfully maintained when isotonic solutions of these divalent cations are used. Fura-2 quenching experiments with Mn(2+), Co(2+), or Ni(2+) suggest that these can be transported by TRPM7 in the presence of physiological levels of Ca(2+) and Mg(2+), suggesting that TRPM7 represents a novel ion-channel mechanism for cellular metal ion entry into vertebrate cells.     

Ion Relations, Chlorophyll Synthesis and the Question of 'Bulk' Phytochrome in Pisum sativum

Lack of correlation between spectrophotometric phytochrome assays and physiological experiments has led to the concept of a ‘bulk’ phytochrome that is measurable but inactive, in contrast to an active form that is not measured in the standard assay. Because of suggestions that phytochrome action involves changes in membrane permeability, particularly to potassium ions, we investigated potassium and sodium uptake and efflux in Pisum sativum epicotyl sections which were equally de-etiolated yet differed three-fold in their phytochrome contents. Although dependent on the metabolic integrity of cell membranes (as shown by respiratory inhibitors) the selective uptake of potassium in this tissue is not under phytochrome control. Experiments were also conducted to see whether a three-fold difference in phytochrome content affects chlorophyll synthesis in Pisum. Differences in chlorophyll synthesis observed can be related to the effectiveness of the pretreatments in eliminating the lag phase, not to their differences in phytochrome; P_fr decay rate has no effect on chlorophyll synthesis. The failure to find any correlation with the amount or state of ‘bulk’ phytochrome in these and previous experiments indicates that no function of this material is yet known.     

Phytochrome, a family of photoreceptors with multiple physiological roles

Abstract. Photoperception by phytochrome is crucially important at many stages of the plant life cycle in allowing adaptation to a changing light environment. Phytochrome is encoded by a family of genes subject to differential expression in response to environmental and developmental factors. Multiple forms of phytochrome exist with, in some cases, identifiably different spectrophotometric, biochemical and physiological characteristics. This article reviews the regulation of development by phytochrome and discusses evidence from physiological studies of wild type, mutant and transgenic plants consistent with the proposal that different members of the phytochrome family have different photosensory roles. We speculate briefly on the possible evolutionary relationship between the different photosensory roles, and suggest approaches towards further elucidation of the nature of phytochrome-mediated photoperception and adaptation to the natural light environment.     

Phytochrome properties and the molecular environment

In vitro data support a scheme of phytochrome phototransformation involving intermediates in a sequential pathway. The fraction of total phytochrome maintained as intermediate under conditions of pigment cycling as well as the rate of the dark reversion of the far red-absorbing (Pfr) to the red-absorbing form of phytochrome (Pr) has been shown to depend on the molecular environment of the phytochrome molecules. Inverse dark reversion of Pr to Pfr has been observed in vitro. These results contribute toward an understanding of the observed paradoxes between physiological experiments and measurements of the amount and state of phytochrome in vivo. The in vivo spectrophotometric assay measures an average of the properties of phytochrome in different cellular environments, whereas a particular physiological response may be controlled by phytochrome molecules in one particular environment. It is therefore possible that all phytochrome is potentially active and triggers specific responses by virtue of its localization.     

Disturbances on delta aminolevulinate dehydratase (ALA-D) enzyme activity by Pb2+, Cd2+, Cu2+, Mg2+, Zn2+, Na+, K+ and Li+: analysis based on coordination geometry and acid-base Lewis capacity

ALA-D (EC 4.2.1.24) is the first cytosolic enzyme in the haem metabolic pathway. Some metals compete with its major cofactor Zn(2+), modifying both enzyme structure and function. Our purpose was to contribute to the understanding of the biochemical role of metals such as Pb(2+), Cd(2+), Cu(2+), Mg(2+), Zn(2+), Na(+), K(+) and Li(+) on ALA-D, using chicken embryos as experimental model. Mg(2+) and Zn(2+) showed enzyme activation in yolk sac membrane (YSM) (113% at 10(-5) M Mg(2+) and from 10(-4) M Zn(2+)), and slight inactivation in liver. Cd(2+) and Cu(2+) caused a non allosteric inhibition in both tissues (100% from 10(-4) M). Surprisingly Pb(2+) was not such a strong inhibitor. Interference of cations during the Schiff base formation in enzymatic catalysis process is explained considering their Lewis acid-base capacity, coordination geometry and electron configuration of valence. Interactions among monovalent cations and biochemical substances are governed chiefly by its electrostatic potential. 0.1 M K(+) and 0.4 M Na(+) produced 30% of enzymatic inhibition by the interference on interactions among the functional subunits. Li(+) activated the YSM enzyme (130% at 10(-5) M) due to a more specific interaction. This study may contribute to elucidate for the first time the possible structural differences between the YSM and liver enzymes from chicken embryo.     

Spectrophotometric Measurements of Phytochrome in vivo and Their Correlation with Photomorphogenic Responses of Phaseolus

Direct in vivo measurements of phytochrome have been made in Phaseolus vulgaris by 2-filter difference spectrophotometry (Ratiospect). All measurements were made at 730 versus 800 nm and it is assumed that the Δ (ΔOD) is directly proportional to the PFR concentration of phytochrome present. Dose response curves were determined for both physiological and spectrophotometric responses for red induction and far-red photoinactivation. For induction, saturation occurs at 100 mj/cm² and for inactivation at 30 mj/cm². The rate of hook opening and the physiological response measured 20 hours after induction are both shown to be directly proportional to the initial amount of PFR present spectrophotometrically. The sensitivity of the tissue correlates well with the absolute amount of phytochrome present, the inner portion of the hook having the maximum concentration of 0.042 Δ (ΔOD)/g fresh weight. If the total reversible phytochrome concentration is reduced by exposure to red light and allowing PFR to decay out of the system the remaining sensitivity of the tissue is shown to be directly correlated with the amount of PR remaining in the tissue. PFR disappears rapidly in the dark at 25°, and is not detectable after 6 hours. There is no indication that PFR reverts in the system to PR. At 4°, PFR does not disappear measurably up to 1 hour and is nearly totally reversible to PR.     

Physiological metal uptake by Nostoc punctiforme

Trace metals are required for many cellular processes. The acquisition of trace elements from the environment includes a rapid adsorption of metals to the cell surface, followed by a slower internalization. We investigated the uptake of the trace elements Co(2+), Cu(2+), Mn(2+), Ni(2+), and Zn(2+) and the non-essential divalent cation Cd(2+) in the cyanobacterium Nostoc punctiforme. For each metal, a dose response study based on cell viability showed that the highest non-toxic concentrations were: 0.5?μM Cd(2+), 2?μM Co(2+), 0.5?μM Cu(2+), 500?μM Mn(2+), 1?μM Ni(2+), and 18?μM Zn(2+). Cells exposed to these non-toxic concentrations with combinations of Zn(2+) and Cd(2+), Zn(2+) and Co(2+), Zn(2+) and Cu(2+) or Zn(2+) and Ni(2+), had reduced growth in comparison to controls. Cells exposed to metal combinations with the addition of 500?μM Mn(2+) showed similar growth compared to the untreated controls. Metal levels were measured after one and 72?h for whole cells and absorbed (EDTA-resistant) fractions and used to calculate differential uptake rates for each metal. The differences in binding and internalisation between different metals indicate different uptake processes exist for each metal. For each metal, competitive uptake experiments using (65)Zn showed that after 72?h of exposure Zn(2+) uptake was reduced by most metals particularly 0.5?μM Cd(2+), while 2?μM Co(2+) increased Zn(2+) uptake. This study demonstrates that N. punctiforme discriminates between different metals and favourably substitutes their uptake to avoid the toxic effects of particular metals.     

Prevention of Action of Far-Red-Absorbing Phytochrome in Rumex crispus L. Seeds by Ethanol

Phytochrome-enhanced germination of curled dock (Rumex crispus L.) seeds is further stimulated by pretreatments in solutions of 0.5 to 2 molar methanol and 0.03 to ≥ 0.3 molar 2-propanol during a 2-day 20°C imbibition. Similar pretreatments in 0.1 molar ethanol, acetaldehyde, and n-propanol inhibit phytochrome-enhanced germination. If exposure to ethanol is delayed until 16 hours after a red irradiation, seeds escape the ethanol inhibition indicating a mechanism other than toxicity. The rate of escape from ethanol inhibition roughly parallels the escape from phytochrome control in seeds held in water only, indicating possible ethanol effects on phytochrome. It was found that ethanol pretreatment prevents the far-red absorbing form of phytochrome (Pfr) from acting but does not accelerate dark decay or prevent transformation. Ethanol inhibition may be prevented if ethanol pretreatment is at 10°C instead of 20°C, or may be overcome by transferring ethanol-pretreated seeds to 10°C in water. Similarly, ethanol inhibition can be overcome by a 2-hour 40°C temperature shift concluding the pretreatment. It is proposed that the ethanol causes perturbations at a membrane which prevent Pfr from acting.     

The ionophore nigericin transports Pb2+ with high activity and selectivity: a comparison to monensin and ionomycin

The K(+) ionophore nigericin is shown to be highly effective as an ionophore for Pb(2+) but not other divalent cations, including Cu(2+), Zn(2+), Cd(2+), Mn(2+), Co(2+), Ca(2+), Ni(2+), and Sr(2+). Among this group a minor activity for Cu(2+) transport is seen, while for the others activity is near or below the limit of detection. The selectivity of nigericin for Pb(2+) exceeds that of ionomycin or monensin and arises, at least in part, from a high stability of nigericin-Pb(2+) complexes. Plots of log rate vs log Pb(2+) or log ionophore concentration, together with the pH dependency, indicate that nigericin transports Pb(2+) via the species NigPbOH and by a mechanism that is predominately electroneutral. As with monensin and ionomycin, a minor fraction of activity may be electrogenic, based upon a stimulation of rate that is produced by agents which prevent the formation of transmembrane electrical potentials. Nigericin-catalyzed Pb(2+) transport is not inhibited by physiological concentrations of Ca(2+) or Mg(2+) and is only modestly affected by K(+) and Na(+) concentrations in the range of 0-100 mM. These characteristics, together with higher selectivity and efficiency, suggest that nigericin may be more useful than monensin in the treatment of Pb intoxication.     

De novo synthesis of phytochrome in pumpkin hooks

Phytochrome becomes density labeled in the hook of pumpkin (Cucurbita pepo L.) seedlings grown in the dark on D₂O, indicating that the protein moiety of the pigment is synthesized de novo during development. Red light causes a rapid decline of the total phytochrome level in the hook of etiolated seedlings but upon return to the dark, phytochrome again accumulates. These newly appearing molecules are also synthesized de novo. Newly synthesized phytochrome in both dark-grown and red-irradiated seedlings is in the red-absorbing form. Turnover of the red-absorbing form is indicated by the density labeling of phytochrome during a period when the total phytochrome level in the hook of dark-grown seedlings remains constant. However, it was not possible to determine whether this results from intracellular turnover or turnover of the whole cell population during hook growth.     

Molecular operation of metals into the function and state of photosystem II

Action sites of different metals in the electron transport reactions of Photosystem II (PS II) evaluated by delayed fluorescence in the ms range (ms DF) and pigment-pigment, pigment-protein and protein-protein interaction states by electrophoretic measurements are presented. The main targets for the metals action were shown to be:(i) Cd(2+), Ni(2+), Co(2+)-Y(z) or CaMn(4)-cluster on the donor site with dependence on pH;(ii) Ni(2+), Co(2+), Zn(2+), Al(3+), Mn(2+) between Q(A) and Q(B) on the acceptor site; effect of Al(3+) and Mn(2+) is observed only in acidic pH. Investigated metals bring about monomerization of oligomeric and dimeric chlorophyll-protein complexes (CPC) and destabilization of protein-protein interactions. Molecular mechanisms of metals interference with the structure of PS II are discussed.     

Inhibition of human serum arylesterase by metal chlorides

The inhibition of arylesterase (paraoxonase, EC 3.1.8.1) by metal chlorides was studied with both pooled human serum (A phenotype) and purified enzyme, using phenyl acetate as substrate. Inhibition data were analysed with the Hill equation. Results obtained with whole serum and purified enzyme were very similar. On the basis of the Hill coefficient, n(H), three groups of inhibitors were distinguished: (1) Cu(2+) and Hg(2+) for which n(H)=1, suggesting a single binding site (probably the free cysteine at position 283); these metals were mixed inhibitors, with more affinity for the free enzyme than for the enzyme-substrate complex; (2) Mn(2+), Co(2+), Ni(2+), Zn(2+), and Cd(2+) for which n(H)>1, suggesting several cooperative binding sites; (3) La(3+), for which n(H)<1. Within groups (1) and (2) the inhibiting potency followed the order of the periodic table. For the 3d elements the inhibiting order followed the Irving-Williams series, with the classical exception of Cu(2+). Only Zn(2+) was inhibitory at its physiological concentration.