Effect of preventive and regressive isosorbide 5-mononitrate treatment on catecholamine levels in plasma, platelets, adrenals, left ventricle and aorta in cyclosporin A-induced hypertensive rats

Increased vascular reactivity associated with cyclosporin A (CsA)-induced arterial hypertension might result from increased vasoconstriction and/or decreased vasodilatation. The administration of organic NO donors could have beneficial effects by the NO-cGMP reposition, but there is the risk of sympathetic nervous system worsening by neuro-hormonal counter-regulation. We evaluate the effect of preventive and regressive (curative) isosorbide 5-mononitrate (Is-5-Mn) treatment on blood pressures and on plasma, platelets, adrenals, left ventricle and aorta norepinephrine (NE) and epinephrine (E) contents, assessed by HPLC, in CsA-induced hypertensive rats. Five rat groups were tested: control (orange juice), CsA (5 mg/kg/day) and Is-5-Mn (150 mg/kg/day, bid) groups were treated for 7 weeks; preventive group (Is-5-Mn+CsA): Is-5-Mn during 2 weeks plus 7 weeks of Is-5-Mn+CsA; regressive group (CsA+Is-5-Mn): CsA during 7 weeks plus 5 weeks of CsA+Is-5-Mn. The increased BP in the CsA group was prevented, but was not reverted, by concomitant Is-5-Mn treatment. In the CsA-treated rats, there was a noticeable decrease in left ventricle NE and E contents and aorta NE levels and a moderate increase in circulating catecholamines, without significant effect in the adrenals values. When Is-5-Mn was preventively used, the CsA-induced effect on left ventricle and aorta was prevented. Concomitantly, however, the plasma-platelet catecholamine balance was disrupted, accumulating NE in plasma, whereas E increased in aorta, mimic the single Is-5-Mn-treated group. In opposition, in the group used as regressive Is-5-Mn therapy, the adrenals contents were higher compared with the CsA-group and, simultaneously, the CsA-evoked effects on circulating, left ventricle and aorta catecholamines were not reverted. In conclusion, regressive Is-5-Mn therapy was unable to attenuate CsA-induced catecholamine changes and BP values even worsened. On the contrary, preventive Is-5-Mn treatment prevented the catecholamine changes on left ventricle and aorta, but increased plasma NE and aorta E accumulation. Even though with those effects, hypertension development was totally prevented, suggesting that peripheral SNS per se cannot fully explain CsA-induced hypertension. Furthermore, Is-5-Mn might produce beneficial effects only if preventively employed but, considering the changes on peripheral catecholamine contents, a judicious evaluation of the nitrate therapy impact is recommended in order to avoid further deleterious effects.     

Bridging-type changes facilitate successive oxidation steps at about 1 V in two binuclear manganese complexes--implications for photosynthetic water-oxidation

The redox behavior of two synthetic manganese complexes illustrates a mechanistic aspect of importance for light-driven water oxidation in Photosystem II (PSII) and design of biomimetic systems (artificial photosynthesis). The coupling between changes in oxidation state and structural changes was investigated for two binuclear manganese complexes (1 and 2), which differ in the set of first sphere ligands to Mn (N(3)O(3) in 1, N(2)O(4) in 2). Both complexes were studied by electron paramagnetic resonance (EPR) and X-ray absorption spectroscopy (XAS) in three oxidation states which had been previously prepared either electro- or photochemically. The following bridging-type changes are suggested. In 1: Mn(II)-(mu-OR)(mu-OCO)(2)-Mn(II)<-->Mn(II)-(mu-OR)(mu-OCO)(2)-Mn(III)-->Mn(III)-(mu-OR)(mu-OCO)(mu-O)-Mn(III). In 2: Mn(II)-(mu-OR)(mu-OCO)(2)-Mn(III)<-->Mn(III)-(mu-OR)(mu-OCO)(2)-Mn(III)-->Mn(III)-(mu-OR)(mu-OCO)(mu-O)-Mn(IV). In both complexes, the first one-electron oxidation proceeds without bridging-type change, but involves a redox-potential increase by 0.5-1V. The second one-electron oxidation likely is coupled to mu-oxo-bridge (or mu-OH) formation which seems to counteract a further potential increase. In both complexes, mu-O(H) bridge formation is associated with a redox transition proceeding at approximately 1V, but the mu-O(H) bridge is observed at the Mn(2)(III,III) level in 1 and at the Mn(III,IV) level in 2, demonstrating modulation of the redox behavior by the terminal ligands. It is proposed that also in PSII bridging-type changes facilitate successive oxidation steps at approximately the same potential.     

Probing the geometric and electronic structures of the low-temperature azide adduct and the product-inhibited form of oxidized manganese superoxide dismutase

The geometric and electronic structures of the six-coordinate azide adduct of oxidized manganese superoxide dismutase (Mn3+ SOD) that is formed at low temperatures, LT N3-Mn3+ SOD, has been examined in detail through a combined spectroscopic/computational approach. Electronic absorption, circular dichroism (CD), magnetic CD (MCD) and variable-temperature, variable-field (VTVH) MCD spectroscopies were used to determine electronic transition energies and to obtain an estimate of zero-field splitting parameters for LT N3-Mn3+ SOD. These experimental data were utilized in conjunction with semiempirical intermediate neglect of differential overlap/spectroscopic parametrization-configuration interaction (INDO/S-CI) and time-dependent density functional theory (TD-DFT) computations to evaluate hypothetical active-site models of LT N3-Mn3+ SOD generated by constrained DFT geometry optimizations. Collectively, our spectroscopic/computational results indicate that N3- binding to Mn3+ SOD at low temperatures promotes neither protonation of the axial solvent ligand nor reorientation of the redox-active molecular orbital, both of which had been previously suggested. Using the same experimentally validated computational approach, models of the product-inhibited form of MnSOD were also developed and evaluated by their relative energies and TD-DFT-computed absorption spectra. On the basis of our computational results as well as previously published kinetic data, we propose that the product-inhibited form of MnSOD is best described as a side-on peroxo-Mn3+ adduct possessing an axial H2O ligand. Notably, attempts to generate a stable hydroperoxo-Mn3+ SOD species by protonation of the proximal O atom of the hydroperoxo ligand resulted in dissociation of HOO- and eventual H+ transfer from Tyr34 to HOO-, generating deprotonated Tyr34 and H2O2. The implications of these results with respect to the mechanism of O2*- dismutation by MnSOD are discussed.     

A carboxylic residue at the high-affinity, Mn-binding site participates in the binding of iron cations that block the site

The role of carboxylic residues at the high-affinity, Mn-binding site in the ligation of iron cations blocking the site [Biochemistry 41 (2000) 5854] was studied, using a method developed to extract the iron cations blocking the site. We found that specifically bound Fe(III) cations can be extracted with citrate buffer at pH 3.0. Furthermore, citrate can also prevent the photooxidation of Fe(II) cations by YZ. Participation of a COOH group(s) in the ligation of Fe(III) at the high-affinity site was investigated using 1-ethyl-3-[(3-dimethylamino)propyl] carbodiimide (EDC), a chemical modifier of carboxylic amino acid residues. Modification of the COOH groups inhibits the light-induced oxidation of exogenous Mn(II) cations by Mn-depleted photosystem II (PSII[-Mn]) membranes. The rate of Mn(II) oxidation saturates at > or = 10 microM in PSII(-Mn) membranes and > or = 500 microM in EDC-treated PSII (-Mn) samples. Intact PSII(-Mn) membranes have only one site for Mn(II) oxidation via YZ (dissociation constant, Kd = 0.64 microM), while EDC-treated PSII(-Mn) samples have two sites (Kd = 1.52 and 22 microM; the latter is the low-affinity site). When PSII(-Mn) membranes were incubated with Fe(II) before modifier treatment (to block the high-affinity site) and the blocking iron cations were extracted with citrate (pH 3.0) after modification, the membranes contained only one site (Kd = 2.3 microM) for exogenous Mn(II) oxidation by Y(Z)() radical. In this case, the rate of electron donation via YZ saturated at a Mn(II) concentration > or = 15 microM. These results indicate that the carboxylic residue participating in Mn(II) coordination and the binding of oxidized manganese cations at the HAZ site is protected from the action of the modifier by the iron cations blocking the HAZ site. We concluded that the carboxylic residue (D1 Asp-170) participating in the coordination of the manganese cation at the HAZ site (Mn4 in the tetranuclear manganese cluster [Science 303 (2004) 1831]) is also involved in the ligation of the Fe cation(s) blocking the high-affinity Mn-binding site.     

Iron-blocking the high-affinity Mn-binding site in photosystem II facilitates identification of the type of hydrogen bond participating in proton-coupled electron transport via YZ

Incubation of Mn-depleted PSII membranes [PSII(-Mn)] with Fe(II) is accompanied by the blocking of Y(Z)(*) at the high-affinity Mn-binding site to exogenous electron donors [Semin et al. (2002) Biochemistry 41, 5854-5864] and a shift of the pK(app) of the hydrogen bond partner for Y(Z) (base B) from 7.1 to 6.1 [Semin, B. K., and Seibert, M. (2004) Biochemistry 43, 6772-6782]. Here we calculate activation energies (E(a)) for Y(Z)(*) reduction in PSII(-Mn) and Fe-blocked PSII(-Mn) samples [PSII(-Mn, +Fe)] from temperature dependencies of the rate constants of the fast and slow components of the flash-probe fluorescence decay kinetics. At pH < pK(app) (e.g., 5.5), the decays are fit with one (fast) component in both types of samples, and E(a) is equal to 42.2 +/- 2.9 kJ/mol in PSII(-Mn) and 46.4 +/- 3.3 kJ/mol in PSII(-Mn, +Fe) membranes. At pH > pK(app), the decay kinetics exhibit an additional slow component in PSII(-Mn, +Fe) membranes (E(a) = 36.1 +/- 7.5 kJ/mol), which is much lower than the E(a) of the corresponding component observed for Y(Z)(*) reduction in PSII(-Mn) samples (48.1 +/- 1.7 kJ/mol). We suggest that the above difference results from the formation of a strong low barrier hydrogen bond (LBHB) between Y(Z) and base B in PSII(-Mn, +Fe) samples. To confirm this, Fe-blocking was performed in D(2)O to insert D(+), which has an energetic barrier distinct from H(+), into the LBHB. Measurement of the pH effects on the rates of Y(Z)(*) reduction in PSII(-Mn, +Fe) samples blocked in D(2)O shows a shift of the pK(app) from 6.1 to 7.6, and an increase in the E(a) of the slow component. This approach was also used to measure the stability of the Y(Z)(*) EPR signal at various temperatures in both kinds of membranes. In PSII(-Mn) membranes, the freeze-trapped Y(Z)(*) radical is stable below 190 K, but half of the Y(Z)(*) EPR signal disappears after a 1-min incubation when the sample is warmed to 253 K. In PSII(-Mn, +Fe) samples, the trapped Y(Z)(*) radical is unstable at a much lower temperature (77 K). However, the insertion of D(+) into the hydrogen bond between Y(Z) and base B during the blocking process increases the temperature stability of the Y(Z)(*) EPR signal at 77 K. Again, these results indicate that Fe-blocking involves Y(Z) in the formation of a LBHB, which in turn is consistent with the suggested existence of a LBHB between Y(Z) and base B in intact PSII membranes [Zhang, C., and Styring, S. (2003) Biochemistry 42, 8066-8076].     

Iron bound to the high-affinity Mn-binding site of the oxygen-evolving complex shifts the pK of a component controlling electron transport via Y(Z)

Flash-probe fluorescence spectroscopy was used to compare the pH dependence of charge recombination between Y(Z)(*) and Q(a)(-) in Mn-depleted, photosystem II membranes [PSII(-Mn)] and in membranes with the high-affinity (HA(Z)) Mn-binding site blocked by iron [PSII(-Mn,+Fe); Semin, B. K., Ghirardi, M. L., and Seibert, M. (2002) Biochemistry 41, 5854-5864]. The apparent half-time for fluorescence decay (t(1/2)) in PSII(-Mn) increased from 9 ms at pH 4.4 to 75 ms at pH 9.0 [with an apparent pK (pK(app)) of 7.1]. The actual fluorescence decay kinetics can be fit to one exponential component at pH <6.0 (t(1/2) = 9.5 ms), but it requires an additional component at pH >6.0 (t(1/2) = 385 ms). Similar measurements with PSII(-Mn,+Fe) membranes show that iron binding has little effect on the maximum and minimum t(1/2) values measured at alkaline and acidic pHs but that it does shift the pK(app) from 7.1 to 6.1 toward the more acidic pK(app) value typical of intact membranes. Light-induced Fe(II) blocking of the PSII(-Mn) membrane is accompanied by a decrease in buffer Fe(II) concentration. This decrease was not the result of Fe(II) binding, but rather of its oxidation at two sites, the HA(Z) site and the low-affinity site. M?ssbauer spectroscopy at 80 K on PSII(-Mn,+Fe) samples, prepared under conditions providing the maximal blocking effect but minimizing the amount of nonspecifically bound iron cations, supports this conclusion since this method detected only Fe(III) cations bound to the membranes. Correlation of the kinetics of Fe(II) oxidation with the blocking parameters showed that blocking occurs after four to five Fe(II) cations were oxidized at the HA(Z) site. In summary, the blocking of the HA(Z) Mn-binding site by iron in PSII(-Mn) membranes not only prevents the access of exogenous donors to Y(Z) but also partially restores the properties of the hydrogen bond net found in intact PS(II), which in turn controls the rate of electron transport to Y(Z).     

Interaction of nitric oxide with the oxygen evolving complex of photosystem II and manganese catalase: a comparative study

Thermus thermophilus catalase. Flash fluorescence studies indicate that the S₃ state of the OEC in the presence of ca. 0.6 mM NO is reduced to the S₁ with an apparent halftime of ca. 0.4 s at about 18 °C, compared with a biphasic decay, with approximate halftimes of 28 s for S₃ to S₂ and 140 s for S₂ to S₁ in the absence of NO. Under similar conditions the S₂ state is reduced by NO to the S₁ state with an approximate halftime of 2 s. These results extend a recent study indicating a slow reduction of the S₁ state at −30°C, via the S₀ and S₋₁ states, to a Mn(II)-Mn(III) state resembling the corresponding state in catalase. The reductive mode of action of NO is repeated with the di-Mn cluster of catalase: the Mn(III)-Mn(III) redox state is reduced to the Mn(II)-Mn(II) state via the intermediate Mn(II)-Mn(III) state. The kinetics of this reduction suggest a decreasing reduction potential with decreasing oxidation state, similar to what is observed with the active states of the OEC. What is unique about the OEC is the rapid interaction of NO with the S₃ state of the OEC, which is compatible with a metalloradical character of this state. Received: 16 June 1999 / Accepted: 28 February 2000     

Kinetics of dissociation of reduced nicotinamide adenine dinucleotide phosphate from its complexes with malic enzyme in relation to substrate inhibition and half-of-the-sites reactivity

Malic enzyme of pigeon liver binds NADPH at four equivalent enzyme sites and binds Mn2+ and malate each at two sets of "tight" and "weak" sites with negative cooperativity [Pry, T. A., & Hsu, R. Y. (1980) Biochemistry 19, 951-962]. Stopped-flow studies on the displacement of NADPH from the malate-enzyme complexes E4-NADPH4, E4-Mn2(2+)-NADPH4, E4-Mn2(2+)-NADPH4-dimalate, and E4-Mn2(2+)-NADPH4-tetramalate by large excess NADP+ or its analogue phosphoadenosine(2')diphospho(5')ribose show that NADPH dissociates from the binary complex rapidly with a first-order rate constant of 427 s-1. Dissociation from the ternary E4-Mn2(2+)-NADPH4 complex containing two tightly bound Mn2+ ions can be described by a single first-order process with a rate constant of 135 s-1, or more satisfactorily by two simultaneous first-order processes attributable to the reactions of Mn2+-deficient (k congruent to 427 s-1) and Mn2+-liganded (k = 96 s-1) subunits. The latter equals twice the maximum steady-state turnover rate of 53.2 + 3.0 s-1 assigned to dissociation of the reduced nucleotide from transient E-Mn2+-NADPH, and this 2:1 ratio strongly supports our proposed "half-of-the-sites" model [Hsu, R. Y., & Pry, T. A. (1980) Biochemistry 19, 962-968]. Dissociation from the E4-Mn2(2+)-NADPH4-dimalate complex (k = 100 s-1) follows only the slower process, suggesting that occupancy of malate at two sites tightens enzyme-bound NADPH on the adjacent sites. Binding of malate at two additional weak sites yields E4-Mn2(2+)-NADPH4-tetramalate and a NADPH dissociation rate constant of 2.69 s-1. The 97% decrease in NADPH dissociation parallels the observed 93% maximal inhibition by malate and is the cause of substrate inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structures of rat cytosolic PEPCK: insight into the mechanism of phosphorylation and decarboxylation of oxaloacetic acid

The structures of the rat cytosolic isoform of phosphoenolpyruvate carboxykinase (PEPCK) reported in the PEPCK-Mn2+, -Mn2+-oxaloacetic acid (OAA), -Mn2+-OAA-Mn2+-guanosine-5'-diphosphate (GDP), and -Mn2+-Mn2+-guanosine-5'-tri-phosphate (GTP) complexes provide insight into the mechanism of phosphoryl transfer and decarboxylation mediated by this enzyme. OAA is observed to bind in a number of different orientations coordinating directly to the active site metal. The Mn2+-OAA and Mn2+-OAA-Mn2+GDP structures illustrate inner-sphere coordination of OAA to the manganese ion through the displacement of two of the three water molecules coordinated to the metal in the holo-enzyme by the C3 and C4 carbonyl oxygens. In the PEPCK-Mn2+-OAA complex, an alternate bound conformation of OAA is present. In this conformation, in addition to the previous interactions, the C1 carboxylate is directly coordinated to the active site Mn2+, displacing all of the waters coordinated to the metal in the holo-enzyme. In the PEPCK-Mn2+-GTP structure, the same water molecule displaced by the C1 carboxylate of OAA is displaced by one of the gamma-phosphate oxygens of the triphosphate nucleotide. The structures are consistent with a mechanism of direct in-line phosphoryl transfer, supported by the observed stereochemistry of the reaction. In the catalytically competent binding mode, the C1 carboxylate of OAA is sandwiched between R87 and R405 in an environment that would serve to facilitate decarboxylation. In the reverse reaction, these two arginines would form the CO2 binding site. Comparison of the Mn2+-OAA-Mn2+GDP and Mn2+-Mn2+GTP structures illustrates a marked difference in the bound conformations of the nucleotide substrates in which the GTP nucleotide is bound in a high-energy state resulting from the eclipsing of all three of the phosphoryl groups along the triphosphate chain. This contrasts a previously determined structure of PEPCK in complex with a triphosphate nucleotide analogue in which the analogue mirrors the conformation of GDP as opposed to GTP. Last, the structures illustrate a correlation between conformational changes in the P-loop, the nucleotide binding site, and the active site lid that are important for catalysis.     

Characteristic features of the interaction between Fe(II) cations and the donor side of the manganese-depleted photosystem II

Ferrous iron cations Fe(II) can effectively bind to the donor side of the manganese-depleted photosystem II (PSII(-Mn)) and in this way block electron transfer from diphenylcarbazide (DPC) to the major donor for P680, Y_Z. The present study was focused on the characteristic features of this process. The oxidation and subsequent binding of Fe(II) cations to PSII(-Mn) may proceed in the absence of an artificial electron acceptor, and therefore we investigated the role of O₂ as a putative endogenous acceptor. Oxygen was shown to participate in the blockade of Y_Z by Fe cations, apparently as a structural element of Fe cluster formed at the donor side of PSII(-Mn). The kinetic study of blocking Y_Z by Fe(II) as dependent on light intensity demonstrated that the quantum efficiency of Fe cations binding to the donor side of PSII(-Mn) considerably exceeded that of Mn cations. We also compared the possibilities of extracting the native Mn cluster and reconstructed Fe cations from PSII and an alternative electron transport from DPC to P680⁺ under the conditions of the Y_Z blockade by Fe cations. Neither an alternative donor for P680, Y_D , nor cytochrome b ₅₅₉ participated in the latter process. As a whole, our evidence shows that many features of binding Fe cation to the donor side of PSII(-Mn) are in common with photoassembling the Mn cluster.Translated from Fiziologiya Rastenii, Vol. 52, No. 1, 2005, pp. 12–20.Original Russian Text Copyright © 2005 by Lovyagina, Davletshina, Kultysheva, Timofeev, Ivanov, Semin.     

Orientation of the Mn(II)-Mn(III) dimer which results from the reduction of the oxygen-evolving complex of photosystem II by NO: an electron paramagnetic resonance study

The central part of the oxygen-evolving complex of photosystem II is a cluster of four manganese atoms. The known EPR spectra in the various oxidation states of the cluster are complicated by the magnetic interactions of the four Mn ions and accordingly are difficult to analyze. It has been shown recently that NO at -30 degrees C slowly reduces the cluster to a Mn(II)-Mn(III) state [Sarrou, J., Ioannidis, N., Deligiannakis, Y., and Petrouleas, V. (1998) Biochemistry 37, 3581-3587). We study herein the orientation dependence of the Mn(II)-Mn(III) EPR spectrum with respect to the thylakoid membrane plane. Both the powder and the oriented spectra are satisfactorily simulated with the same set of fine and hyperfine parameters assuming axial symmetry and collinear g and A tensors. The axial component of the tensors is found to be oriented at an angle of 20 degrees +/- 10 degrees to the membrane plane normal (mosaic spread Omega = 40 degrees ). We make the reasonable assumption that the Mn(II)-Mn(III) dimer is one of the di-mu-oxo units that has been suggested to comprise the Mn tetramer. On the basis of the sign of the hyperfine tensor anisotropy, the axial direction is assigned to the d(z(2)) orbital of Mn(III), which by comparison with synthetic model complexes is assumed to be oriented perpendicular to the Mn-(mu-oxo)-Mn plane. The present results complement earlier orientation studies by EXAFS and suggest that the Mn-(mu-oxo)-Mn plane makes a small angle (approximately 20 degrees) with the membrane plane and the axis connecting the bridging oxygens is approximately parallel to the plane.     

Amphiphilic corroles bind tightly to human serum albumin

Amphiphilic 2,17-bis-sulfonato-5,10,15(trispentafluorophenyl)corrole (2) and its Ga and Mn complexes (2-Ga and 2-Mn) form tightly bound noncovalent conjugates with human serum albumin (HSA). Protein-induced changes in the electronic absorption, emission, and circular dichroism spectra of these corroles, as well as results obtained from HPLC profiles of the conjugates and selective fluorescence quenching of the single HSA tryptophan, are interpreted in terms of multiple corrole:HSA binding sites. High-affinity binding sites, close to the unique tryptophan, are fully occupied at very low concentrations. At biologically relevant HSA concentrations (2-3 orders of magnitude larger than those employed in our studies), all corroles (2, 2-Ga, and 2-Mn) may be considered as fully conjugated.     

Structural definition of the active site and catalytic mechanism of 3,4-dihydroxy-2-butanone-4-phosphate synthase

X-ray crystal structures of L-3,4-dihydroxy-2-butanone-4-phosphate synthase from Magnaporthe grisea are reported for the E-SO(4)(2-), E-SO(4)(2-)-Mg(2+), E-SO(4)(2)(-)-Mn(2+), E-SO(4)(2)(-)-Mn(2+)-glycerol, and E-SO(4)(2)(-)-Zn(2+) complexes with resolutions that extend to 1.55, 0.98, 1.60, 1.16, and 1.00 A, respectively. Active-site residues of the homodimer are fully defined. The structures were used to model the substrate ribulose 5-phosphate in the active site with the phosphate group anchored at the sulfate site and the placement of the ribulose group guided by the glycerol site. The model includes two Mg(2+) cations that bind to the oxygen substituents of the C2, C3, C4, and phosphate groups of the substrate, the side chains of Glu37 and His153, and water molecules. The position of the metal cofactors and the substrate's phosphate group are further stabilized by an extensive hydrogen-bond and salt-bridge network. On the basis of their proximity to the substrate's reaction participants, the imidazole of an Asp99-His136 dyad from one subunit, the side chains of the Asp41, Cys66, and Glu174 residues from the other subunit, and Mg(2+)-activated water molecules are proposed to serve specific roles in the catalytic cycle as general acid-base functionalities. The model suggests that during the 1,2-shift step of the reaction, the substrate's C3 and C4 hydroxyl groups are cis to each other. A cis transition state is calculated to have an activation barrier that is 2 kcal/mol greater than that of the trans transition state in the absence of the enzyme.     

Corroles that bind with high affinity to both apo and holo transferrin

The interactions of transferrin (Tf) with the water soluble corrole 1 and with its gallium (1-Ga) and manganese (1-Mn) complexes were studied to establish the possible utilization of corrole-transferrin conjugates for targeting these corroles to cells that express the transferrin receptor. The protein, in both its iron-free apo form (apoTf) and the iron-bound holo form (holoTf), was found to spontaneously bind all three derivatives. This conclusion was reached from titrations followed by several spectroscopic methods and dilution experiments measured by fluorescence. The such elucidated very small dissociation constant of 2 x 10(-7) M and 3 x 10(-8) M for 1-Ga with apoTf and holoTf, respectively and <10(-9) M for 1 with both protein forms are clearly relevant for the physiological concentration of transferrin in serum.     

Proton and deuteron nuclear magnetic relaxation dispersion studies of Ca2+-Mn2+-lentil lectin and Ca2+-Mn2+-pea lectin: evidence for a site of solvent exchange in common with concanavalin A

Measurements of the magnetic field dependence of the longitudinal magnetic relaxation rates (NMRD profiles) of solvent protons and deuterons led to the discovery of two classes of solvent binding sites in Ca2+-Mn2+-concanavalin A (CMPL) [Koenig, S. H., Brown, R. D., III, & Brewer, C. F. (1985) Biochemistry (second of three papers in this issue)]. In this paper, we compare proton and deuteron NMRD profiles of Ca2+-Mn2+-lentil lectin (CMLcH) and Ca2+-Mn2+-pea lectin (CMPSA) with those of CMPL. All three metalloproteins are D-mannose/D-glucose-specific lectins that have a high degree of structural similarity and require the metal ions for their biological activities. We have developed a method for the preparation of fully active metal ion derivatives of lentil lectin (LcH) and pea lectin (PSA), including the diamagnetic derivatives Ca2+-Zn2+-LcH and Ca2+-Zn2+-PSA [Bhattacharyya, L., Brewer, C. F., Brown, R. D., III, & Koenig, S. H.(1984) Biochem. Biophys. Res. Commun. 124, 857-862]. The behavior of these two lectins with regard to their NMRD profiles is essentially identical, for both the paramagnetic and diamagnetic forms. Together with CMPL, all three lectins have a common paramagnetic contribution with a negative temperature dependence of the rates, while CMPL contributes an additional component with a positive temperature dependence. The common contribution derives from the class of fast exchanging water molecules observed in the proton NMRD profile of CMPL (Koenig et al., 1985); their protons are calculated to be relatively remote from the Mn2+ ions (4.4 A for CMPL and 5.5 A for LcH and PSA).(ABSTRACT TRUNCATED AT 250 WORDS)     

DNA cleavage by homo- and heterotetranuclear Cu(II) and Mn(II) complexes with tetrathioether-tetrathiol moiety

Novel homotetranuclear Cu(II) and heteronuclear Cu(II)-Mn(II) complexes with tetrathioether-tetrathiol moiety have been prepared and their DNA relaxation activities with plasmid pCYTEXP (5kb) were electrophoretically established. The cleavage products analyzed by neutral agarose gel electrophoresis indicated that the interaction of the metal complexes with supercoiled plasmid DNA yielded linear, nicked or degraded DNA. The relaxation activities of both homo- and heterotetranuclear (SK4) complexes are time- and concentration-dependent. The findings suggest that SK4 with potent nucleolytic activity is a good nuclease substitute in the presence of cooxidant. Furthermore, the observation of induction of DNA into smaller fragments by SK4 is also significant.     

Effects of iron-, manganese-, or magnesium-deficiency on the growth and morphology of Euglena gracilis

Iron-, manganese-, or magnesium-deficiency has been induced in Euglena gracilis. Each arrests cell proliferation, decreases the intracellular content of the deficient metal, and increases that of several other metals. Light and electron microscopy of stationary phase cells reveal that Fe-deficient (-Fe) cells are similar in size and shape to control organisms. Magnesium-deficient (-Mg) cells, however, are larger, and approximately 14% are multilobed, containing 2 to 12 lobes of equal size emanating from a central region. Individual (-Mg) cells and each lobe of multilobed cells contain a single nucleus. Manganese-deficient (-Mn) organisms are morphologically more heterogeneous than (-Fe) or (-Mg) cells. Most are spherical and larger than controls. Approximately 15% are multilobed but, unlike (-Mg) cells, contain lobes of unequal size with either zero, one, or several nuclei present in each. Nuclei of (-Mn) cells differ in size and shape from those of control, (-Fe), or (-Mg) cells. All three deficient cell types accumulate large quantities of paramylon. Other cytoplasmic structures, however, appear normal. Addition of Fe, Mn, or Mg to the respective deficient stationary phase cultures reverses growth arrest and restores normal morphology. The results suggest that Fe-, Mn-, and Mg-deficiencies affect different stages of the E. gracilis cell cycle.     

Oxidative cleavage of DNA by homo- and heteronuclear Cu(II)-Mn(II) complexes of an oxime-type ligand

Novel homodinuclear Cu(II) (K1), heterodinuclear Cu(II)-Mn(II) (K2) and homotrinuclear Cu(II) (K3) complexes with a novel oxime-type ligand have been prepared and their nucleolytic activities on pCYTEXP were established by neutral agarose gel electrophoresis. The analyses of the cleavage products obtained electrophoretically indicate that although the examined complexes induces very similar conformational changes on supercoiled DNA by converting supercoiled form to nicked form than linear form in a sequential manner as the complex concentration or reaction period is increased, K3 is less effective than the two others. The oxime complexes were nucleolytically active at physiological pH values but the activities of K1 or K2 were diminished by increasing the pH of the reaction mixture. In contrast, K3 makes dominantly single strand nicking by producing nicked circles on DNA at almost all the applied pH values. Metal complex induced DNA cleavage was also tested for inhibition by various radical scavengers as superoxide dismutase (SOD), azide, thiourea and potassium iodide. The antioxidants inhibited the nucleolytic acitivities of the oxime complexes but SOD afforded no protection indicating that the nucleolytic mechanism involves of copper and/or manganese complex-mediated reactive oxygen species such as hydroxyl radicals being responsible for the oxidative DNA cleavage.     

Effects of Iron-, Manganese-, or Magnesium-Deficiency on the Growth and Morphology of Euglena gracilis1

Iron-, manganese-, or magnesium-deficiency has been induced in Euglena gracilis. Each arrests cell proliferation, decreases the intracellular content of the deficient metal, and increases that of several other metals. Light and electron microscopy of stationary phase cells reveal that Fe-deficient (-Fe) cells are similar in size and shape to control organisms. Magnesium-deficient (-Mg) cells, however, are larger, and approximately 14% are multilobed, containing 2 to 12 lobes of equal size emanating from a central region. Individual (-Mg) cells and each lobe of multilobed cells contain a single nucleus. Manganese-deficient (-Mn) organisms are morphologically more heterogeneous than (-Fe) or (-Mg) cells. Most are spherical and larger than controls. Approximately 15% are multilobed but, unlike (-Mg) cells, contain lobes of unequal size with either zero, one, or several nuclei present in each. Nuclei of (-Mn) cells differ in size and shape from those of control, (-Fe), or (-Mg) cells. All three deficient cell types accumulate large quantities of paramylon. Other cytoplasmic structures, however, appear normal. Addition of Fe, Mn, or Mg to the respective deficient stationary phase cultures reverses growth arrest and restores normal morphology. The results suggest that Fe-, Mn-, and Mg-deficiencies affect different stages of the E. gracilis cell cycle.     

Proton and deuteron nuclear magnetic relaxation dispersion studies of Ca2+-Mn2+-concanavalin A: evidence for two classes of exchanging water molecules

We have measured the magnetic field dependence of the nuclear magnetic relaxation rates (NMRD profiles) of solvent protons and deuterons in solutions of Ca2+-Mn2+-concanavalin A (Con A) with and without saccharide present. Data were obtained over the range -8 to 35 degrees C; the extension to the lowest temperature was made possible by the presence of 5 M salt. Since previous theoretical analyses, using accepted relaxation theories of 1H NMRD profiles alone, led to unsatisfactory conclusions, we have attempted to take advantage of the fact that the residence lifetime of a water ligand of the metal ions can influence the relaxation behavior of protons and deuterons differently. From a comparison of the present proton and deuteron results, we find that Ca2+-Mn2+-Con A has two classes of binding sites: one, associated with the inner coordiation sphere of the Mn2+ ions, having a resident lifetime for solvent water of approximately 10(-5) s that is reduced by the presence of saccharide and another having a lifetime of approximately 5 X 10(-9) s, located with the protons of the bound waters approximately 4.4 A from the Mn2+ ions (assuming two equivalent water molecules in this class), which is well beyond the coordination environment of the Mn2+ ions. The relaxation contribution of these more distant sites is unaffected by saccharide. The conclusions are corroborated by measurements of the temperature dependences of the proton NMRD profiles, which show quite clearly that the profiles are composite, containing two contributions with opposite dependences on temperature.(ABSTRACT TRUNCATED AT 250 WORDS)