Manganese-dependent propagated action potentials and their depression by electrical stimulation in guinea-pig myocardium perfused by sodium-free media.
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1. Propagated action potentials were recorded in right ventricular papillary muscles from guinea-pig heart while exposed to Na-free, Ca-free and Mg-free solutions containing Mn. 2. When Na was totally replaced by 95 mM-Mn the overshoot was about 45 mV while the resting potential was about -90mV. 3. The overshoot of action potentials was increased by about 20-30 mV per tenfold increase of Mn concentration over the range of 2-50 mM. 4. Similar increases of overshoots with increasing of Mn concentration also occurred in the presence of 0-6 mM-Ca. Increasing of Ca from 5 to 20 mM had little influence on the overshoot but shortened the duration of the Mn-dependent action potential in the presence of 5 mM-MN. 5 Mn-dependent action potentials were not depressed by 3 X 10(5) M tetrodotoxin but by La. 6. These results suggest that Mn passes through the slow inward current channel to generate the action potential seen under the Na-free condition. 7. The overshoot and duration of the Mn-dependent action potential decreased with stimulation. At stimulus frequencies (Hz) of 0-5, 0-2, 0-1, 0-017 and 0-0033 the overshoot of action potential in 5 mM-Mn Tyrode decreased by 0-5-1 mV per an action potential. This depression of the action potential is explained by assuming intracellular accumulation of Mn. (+info)
Description of a versatile peroxidase involved in the natural degradation of lignin that has both manganese peroxidase and lignin peroxidase substrate interaction sites.
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Two major peroxidases are secreted by the fungus Pleurotus eryngii in lignocellulose cultures. One is similar to Phanerochaete chrysosporium manganese-dependent peroxidase. The second protein (PS1), although catalyzing the oxidation of Mn2+ to Mn3+ by H2O2, differs from the above enzymes by its manganese-independent activity enabling it to oxidize substituted phenols and synthetic dyes, as well as the lignin peroxidase (LiP) substrate veratryl alcohol. This is by a mechanism similar to that reported for LiP, as evidenced by p-dimethoxybenzene oxidation yielding benzoquinone. The apparent kinetic constants showed high activity on Mn2+, but methoxyhydroquinone was the natural substrate with the highest enzyme affinity (this and other phenolic substrates are not efficiently oxidized by the P. chrysosporium peroxidases). A three-dimensional model was built using crystal models from four fungal peroxidase as templates. The model suggests high structural affinity of this versatile peroxidase with LiP but shows a putative Mn2+ binding site near the internal heme propionate, involving Glu36, Glu40, and Asp181. A specific substrate interaction site for Mn2+ is supported by kinetic data showing noncompetitive inhibition with other peroxidase substrates. Moreover, residues reported as involved in LiP interaction with veratryl alcohol and other aromatic substrates are present in peroxidase PS1 such as His82 at the heme-channel opening, which is remarkably similar to that of P. chrysosporium LiP, and Trp170 at the protein surface. These residues could be involved in two different hypothetical long range electron transfer pathways from substrate (His82-Ala83-Asn84-His47-heme and Trp170-Leu171-heme) similar to those postulated for LiP. (+info)
Molecular cloning and characterization of a cDNA for an iron-superoxide dismutase in rice (Oryza sativa L.).
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We have isolated a cDNA encoding Fe-SOD from rice (Oryza sativa L.). The deduced amino acid sequence consists of a polypeptide with 255 amino acids, including a putative transit peptide (40 a.a.) in amino-terminal residues. This sequence is similar to the known plant Fe-SODs but not classified in the group of known Fe-SODs. The metal analysis and SOD assays of the partial purified recombinant protein expressed in E. coli showed that this cDNA encodes an iron-containing SOD. However this SOD activity was not inhibited by the treatment with hydrogen peroxide, which was expected to inhibit known Fe-SOD activity. mRNA of rice Fe-SOD was detected in all vegetative tissues examined, being especially abundant in calli, and strongly increased by light induction. These results suggested that this cDNA encodes rice Fe-SOD, which is apparently distinct from known plant Fe-SODs. (+info)
The titrations of Asp-85 and of the cation binding residues in bacteriorhodopsin are not coupled.
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An outstanding problem relating to the structure and function of bacteriorhodopsin (bR), which is the only protein in the purple membrane of the photosynthetic microorganism Halobacterium salinarium, is the relation between the titration of Asp-85 and the binding/unbinding of metal cations. An extensively accepted working hypothesis has been that the two titrations are coupled, namely, protonation of Asp-85 (located in the vicinity of the retinal chromophore) and cation unbinding occur concurrently. We have carried out a series of experiments in which the purple blue equilibrium and the binding of Mn2+ ions (monitored by electron spin resonance) were followed as a function of pH for several (1-4) R = [Mn2+]/[bR] molar ratios. Data were obtained for native bR, bR mutants, artificial bR and chemically modified bR. We find that in the native pigment the two titrations are separated by more than a pKa unit [delta pKa = pKa(P/B)-pKa(Mn2+) = (4.2-2.8) = 1.4]. In the non-native systems, delta pKa values as high as 5 units, as well as negative delta pKas, are observed. We conclude that the pH titration of cation binding residues in bR is not directly related to the titration of Asp-85. This conclusion is relevant to the nature of the high affinity cation sites in bR and to their role in the photosynthetic function of the pigment. (+info)
31P nuclear magnetic resonance study of the flavoprotein component of the Escherichia coli sulfite reductase.
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SiR-FP60, the monomeric form of the Escherichia coli sulfite reductase flavoprotein component (SiR-FP), has been analysed by 31P-NMR spectroscopy. This protein was reported previously as a reliable simplified model for native SiR-FP [Zeghouf, M., Fontecave, M., Macherel, D., & Coves, J. (1998) Biochemistry 37, 6117-6123]. SiR-FP60 was examined in its native form, as a complex with NADP+ and after monoelectronic reduction either with NADPH or dithionite. In these latter cases, the stabilized FMN semiquinone radical offers a natural and internal paramagnetic probe. The paramagnetic effect of added manganese was also studied. In each case, the NMR parameters were extracted from digitalized data by a deconvolution procedure and compared with those obtained previously with cytochrome P450 reductase. Evolution of the NMR parameters and of calculated relaxation rate constants upon biochemical modifications of SiR-FP60 led us to propose that the reactive center is more compact than the one of cytochrome P450 reductase, with the redox components, FMN, FAD and NADPH, in a tighter spatial arrangement, close to the protein surface. This underlies some subtle differences between the two proteins for which a very similar overall structure is likely considering their common genetic origin and common operating cycle. (+info)
Molecular mechanisms of zinc-dependent leukocyte adhesion involving the urokinase receptor and beta2-integrins.
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The trace element Zinc (Zn2+) has been implicated as a mediator in host defense, yet the molecular basis for its extracellular functions remains obscure. Here, we demonstrate that Zn2+ can induce the adhesion of myelomonocytic cells to the endothelium, as well as to the provisional matrix proteins vitronectin (VN) and fibrinogen (FBG), which are pivotal steps for the recruitment of leukocytes into inflamed/injured tissue. Physiologic concentrations of Zn2+ increased the urokinase receptor (uPAR)-mediated adhesion of myelomonocytic cells to VN, whereas other divalent cations had smaller effects. Zn2+-induced cell adhesion to VN was abolished by cation chelators such as 1-10-phenanthroline, as well as by plasminogen activator inhibitor-1 (PAI-1) and a monoclonal antibody (MoAb) against uPAR. These characteristics could be recapitulated with a uPAR-transfected cell line emphasizing the specificity of this receptor system for Zn2+-dependent cell adhesion. Like urokinase (uPA), Zn2+ increased the binding of radiolabeled VN to uPAR-expressing cells, as well as the interaction of VN with immobilized uPAR in an isolated system. Moreover, Zn2+ enhanced leukocytic cell adhesion to FBG and endothelial cell monolayers by activating beta2-integrins. Instead of the direct beta2-integrin activation through the divalent cation binding site, Zn2+-induced integrin activation was mediated via uPAR, a crucial regulator of this system. The present study uncovers for the first time Zn2+-mediated cell adhesion mechanisms that may play a crucial role in modulating leukocyte adhesion to vessel wall components. (+info)
Direct metal analyses of Mn2+-dependent and -independent protein phosphatase 2A from human erythrocytes detect zinc and iron only in the Mn2+-independent one.
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A Mn2+-dependent protein phosphatase 2A which is composed of a 34 kDa catalytic C' subunit and a 63 kDa regulatory A' subunit, was purified from human erythrocyte cytosol. C' and A' produced V8- and papain-peptide maps identical to those of the 34 kDa catalytic C and the 63 kDa regulatory A subunits of the Mn2+-independent conventional protein phosphatase in human erythrocyte cytosol, respectively. Reconstitution of C'A and CA' revealed that the metal dependency resided in C' and not in A'. In CA, 0.87 +/- 0.12 mol zinc and 0.35 +/- 0.18 mol iron per mol enzyme were detected by atomic absorption spectrophotometry, but manganese, magnesium and cobalt were not detected. None of these metals was detected in C'A'. Pre-incubation of C' with ZnCl2 and FeCl2, but not FeCl3, synergistically stimulated the Mn2+-independent protein phosphatase activity. The protein phosphatase activity of C was unaffected by the same zinc and/or iron treatment. These results suggest that C is a Zn2+- and Fe2+-metalloenzyme and that C' is the apoenzyme. (+info)
Dietary copper, manganese and iron affect the formation of aberrant crypts in colon of rats administered 3,2'-dimethyl-4-aminobiphenyl.
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Aberrant crypt foci (ACF) are preneoplastic lesions for colon cancer. Altered amounts of copper-zinc (CuZnSOD) and manganese (MnSOD) superoxide dismutases have been implicated in multistage carcinogesis of both rodents and humans. Dietary factors are potential modulators of both CuZnSOD and MnSOD activity. The purpose of this study was to investigate the interactive effects of dietary copper, manganese, and iron on 3,2'-dimethyl-4-aminobiphenyl (DMABP)-induced ACF and superoxide dismutase activities in weanling rats fed low or adequate copper (0.8 or 5.1 microg Cu/g diet), low or adequate manganese (0.6 or 17 microg Mn/g diet), and adequate or high iron (37 or 140 microg Fe/g diet). Twelve rats were allowed free access to each of these eight diets for 3.5 wk prior to DMABP administration and for an additional 8 wk after the first DMABP injection. Rats fed low dietary copper had 105% (P < 0.0001) higher formation of DMABP-induced ACF than those fed adequate dietary copper. Rats ingesting low rather than adequate dietary manganese had 23% higher formation of ACF, and rats ingesting high rather than adequate dietary iron had 18% higher formation of ACF. Heart total superoxide dismutase activity was significantly correlated with the number of ACF (r = -0.43, P < 0.0001) in rats administered DMABP. These results suggest that dietary alterations that affect superoxide dismutase activity may affect cancer susceptibility. (+info)