Effect of nucleotides on the oligomeric state of human lactoferrin

Lactoferrin (LF) is a multifunctional acute-phase protein involved in nonspecific defense against bacteria, viruses, and cancer diseases and is present in human barrier fluids, blood, and milk. Small-angle X-ray scattering (SAXS) and light scattering (LS) demonstrated for the first time that LF occurs in the form of oligomers, with a high monomer unit number in the solution. The degree of LF oligomerization depends on the LF concentration and the storage period of non-frozen neutral LF solutions. The average inertial radius of scattering particles (R _g) reaches 100–450 Å at LF concentrations comparable with those in human milk, while R _g of LF monomers is 26.7 Å. LF forms complexes with various nucleotides and hydrolyzes them. The addition of ATP or AMP to LF solutions accelerates LF oligomerization and increases R _g to 600–700 Å, regardless of the initial degree of LF oligomerization. According to the different models (sphere, plate, and cylinder) of LF aggregates, its complexes with such R _g presumably contain several tens to thousands of LF monomers. The possible role of oligomeric complexes in multiple biological functions of LF is discussed.     

Prophylactic effect of human lactoferrin against Streptococcus mutans bacteremia in lactoferrin knockout mice

Streptococcus mutans is the primary agent of dental caries, which is often detected in transient bacteremia. Lactoferrin is a multifunctional glycoprotein showing antibacterial activities against several Streptococcus species. We reported here the prophylactic effect of human lactoferrin (hLF) in a lactoferrin knockout mouse (LFKO^−/−) bacteremic model. The hLF treatment significantly cleared S. mutans from the blood and organs of bacteremic mice when compared to the non-hLF treated mice. Further, analysis of serum cytokines, spleen and liver cytokine mRNA levels revealed that hLF prophylaxis modulates their release differently when compared to the non-hLF treated group. C-reactive protein level (P = 0.003) also decreased following hLF prophylaxis in S. mutans induced bacteremic mice. Additional quantitative RT-PCR analysis revealed that hLF prophylaxis significantly decreased the expression level of IFN-γ, TNF-α, IL-1β, IL-6, MPO and iNOS in spleen and liver. These results suggested that the hLF protects the host against S. mutans-induced experimental bacteremia.     

Fungicidal effect of human lactoferrin against Candida albicans

Human lactoferrin (LF) in its iron-free state (apo LF), killed Candida albicans in a time- and dose-dependent way. The lethal effect was stronger at pH 7.0 than at pH 5.5 and maximum inhibition at neutral pH was achieved in 25 min when the fungal cells were exposed to LF in 0.05 mM KCl at 37 degrees C. Fe(3+)-saturated LF had no fungicidal activity. Apo LF-mediated killing was also temperature-dependent with enhanced inhibition at higher temperatures (37 degrees, 42 degrees C). The presence of 1 mM D-glucose did not affect the candidacidal activity of apo LF but both phosphate and bicarbonate ions at physiological salivary concentrations completely blocked the anti-fungal effect. Therefore it seems unlikely that LF belongs to the major host defence factors against oral candidosis.     

Bactericidal effect of lactoferrin on Legionella pneumophila: effect of the physiological state of the organism

Lactoferrin has been previously shown to be bactericidal for Legionella pneumophila. The current study showed that CaCl2, Mg(NO3)2, and MgCl2, but not NaCl, blocked killing. Activity was pH dependent with the greatest activity at 5.0. Sensitivity of the organism was dramatically affected by the growth conditions. Log phase 12 h, broth-grown cells were most sensitive, with older cultures becoming more resistant. Plate-grown cells were completely resistant. Lactoferrin binding, as detected by immunofluorescence microscopy, was temperature dependent (no binding at 4 degrees C), but was independent of killing.     

Modifying the oligomeric state of cyclic amidase and its effect on enzymatic catalysis

A group of cyclic amidases, including hydantoinase, allantoinase, dihydropyrimidinase, and dihydroorotase, catalyze the reversible hydrolysis of cyclic ureides, such as 5-monosubstituted hydantoins and dihydropyrimidines. These four enzymes carry hydrophobic patches to form dimers. With the exception of dihydroorotase, these enzymes are further dimerized to form tetramers by hydrophobic interactions. This leads us to speculate that the hydrophobic interaction domain may be a significant factor in the catalytic property of these oligomeric cyclic amidases, for which activities are not allosterically regulated. We generated a dimeric D-hydantoinase by mutating five residues in the hydrophobic alpha-helical interface of a tetramer and analyzed the kinetic properties of the dimeric form of D-hydantoinase. The specific activity of the dimeric D-hydantoinase corresponds to 5.3% of the activity of tetrameric D-hydantoinase. This low specific activity of the dimeric D-hydantoinase indicates that the dimeric interaction to form a tetramer has a significant effect on the catalytic activity of this non-allosteric tetramer.     

Redox and metal-regulated oligomeric state for human porphobilinogen synthase activation

The oligomeric state of human porphobilinogen synthase (PBGS) [EC.4.2.1.24] is homooctamer, which consists of conformationally heterogenous subunits in the tertiary structure under air-saturated conditions. When PBGS is activated by reducing agent with zinc ion, a reservoir zinc ion coordinated by Cys²²³ is transferred in the active center to be coordinated by Cys¹²², Cys¹²⁴, and Cys¹³² (Sawada et al. in J Biol Inorg Chem 10:199–207, 2005). The latter zinc ion serves as an electrophilic catalysis. In this study, we investigated a conformational change associated with the PBGS activation by reducing agent and zinc ion using analytical ultracentrifugation, negative staining electron microscopy, native PAGE, and enzyme activity staining. The results are in good agreement with our notion that the main component of PBGS is octamer with a few percent of hexamer and that the octamer changes spatial subunit arrangement upon reduction and further addition of zinc ion, accompanying decrease in f/f ₀. It is concluded that redox-regulated PBGS activation via cleavage of disulfide bonds among Cys¹²², Cys¹²⁴, and Cys¹³² and coordination with zinc ion is closely linked to change in the oligomeric state.     

Reversible denaturation of oligomeric human chaperonin 10: denatured state depends on chemical denaturant

Chaperonins cpn60/cpn10 (GroEL/GroES in Escherichia coli) assist folding of nonnative polypeptides. Folding of the chaperonins themselves is distinct in that it entails assembly of a sevenfold symmetrical structure. We have characterized denaturation and renaturation of the recombinant human chaperonin 10 (cpn10), which forms a heptamer. Denaturation induced by chemical denaturants urea and guanidine hydrochloride (GuHCl) as well as by heat was monitored by tyrosine fluorescence, far-ultraviolet circular dichroism, and cross-linking; all denaturation reactions were reversible. GuHCl-induced denaturation was found to be cpn10 concentration dependent, in accord with a native heptamer to denatured monomer transition. In contrast, urea-induced denaturation was not cpn10 concentration dependent, suggesting that under these conditions cpn10 heptamers denature without dissociation. There were no indications of equilibrium intermediates, such as folded monomers, in either denaturant. The different cpn10 denatured states observed in high [GuHCl] and high [urea] were supported by cross-linking experiments. Thermal denaturation revealed that monomer and heptamer reactions display the same enthalpy change (per monomer), whereas the entropy-increase is significantly larger for the heptamer. A thermodynamic cycle for oligomeric cpn10, combining chemical denaturation with the dissociation constant in absence of denaturant, shows that dissociated monomers are only marginally stable (3 kJ/mol). The thermodynamics for co-chaperonin stability appears conserved; therefore, instability of the monomer could be necessary to specify the native heptameric structure.     

Correlation of biochemical properties with the oligomeric state of human rad52 protein

The human Rad52 protein self-associates to form ring-shaped oligomers, as well as higher order complexes of these rings. We have shown previously that there are two experimentally separable self-association domains in HsRad52, one in the N terminus (residues 1-192) responsible for assembly of individual subunits into rings, and one in the C terminus (residues 218-418) responsible for higher order oligomerization of rings. Earlier studies suggest that the higher order complexes promote DNA end-joining, and others suggest that these complexes are relevant to in vivo Rad52 function. In this study we demonstrate that although inherent binding to single-stranded DNA depends on neither higher order complexes of Rad52 rings nor the ring-shaped oligomers themselves, higher order complexes are important for activities involving simultaneous interaction with more than one DNA molecule. This provides biochemical support for what may be an important in vivo function of Rad52.     

The oligomeric conformation of peroxiredoxins links redox state to function

Protein-protein associations, i.e. formation of permanent or transient protein complexes, are essential for protein functionality and regulation within the cellular context. Peroxiredoxins (Prx) undergo major redox-dependent conformational changes and the dynamics are linked to functional switches. While a large number of investigations have addressed the principles and functions of Prx oligomerization, understanding of the diverse in vivo roles of this conserved redox-dependent feature of Prx is slowly emerging. The review summarizes studies on Prx oligomerization, its tight connection to the redox state, and the knowledge and hypotheses on its physiological function in the cell as peroxidase, chaperone, binding partner, enzyme activator and/or redox sensor.     

The oligomeric state and arrangement of the active bacterial translocon

Protein secretion in bacteria is driven through the ubiquitous SecYEG complex by the ATPase SecA. The structure of SecYEG alone or as a complex with SecA in detergent reveal a monomeric heterotrimer enclosing a central protein channel, yet in membranes it is dimeric. We have addressed the functional significance of the oligomeric status of SecYEG in protein translocation using single molecule and ensemble methods. The results show that while monomers are sufficient for the SecA- and ATP-dependent association of SecYEG with pre-protein, active transport requires SecYEG dimers arranged in the back-to-back conformation. Molecular modeling of this dimeric structure, in conjunction with the new functional data, provides a rationale for the presence of both active and passive copies of SecYEG in the functional translocon.     

The effect of different hormonal conditions on the concentration and oxidoreduction state of the nicotinamide nucleotides of rat liver

1. A method is described for the determination of the oxidized and reduced forms of the nicotinamide nucleotides by measuring the rate of the oxygen uptake with an oxygen electrode in a system in which the nucleotide acts as the rate-limiting carrier in a cyclic system. 2. The method permits the measurement of quantities as low as 0·02μg. of NAD⁺ or NADH or 0·01μg. of NADP⁺ or NADPH. 3. The method permits the measurement of the nucleotides in extracts that contain non-specific reducing substances, coloured compounds or fluorescent materials, e.g. green leaves. 4. The results obtained by the present method are compared with those reported in the literature.     

Inhibitory effect of polyelectrolytes on oligomeric enzymes

The effect of polyelectrolytes on the stability and catalytic characteristics of oligomeric enzymes--pig muscle lactate dehydrogenase (LDH) and bovine liver glutamate dehydrogenase (GDH)--was studied by fluorescent spectroscopic and steady state kinetic methods. It was shown that the binding of negatively charged polyelectrolytes--polystyrene sulfonate, polymethacrylate, and polyphosphate--destroys the tertiary and partially the secondary structure of LDH and GDH, resulting in their complete inactivation at pH < 7. The concentrations of polyelectrolytes needed for inhibition of the enzymes were in this case by two or more orders of magnitude lower than the corresponding concentrations for monomers--toluene sulfonate, methacrylate, and phosphate. The affinity of the substrate (pyruvate) for LDH did not vary in the presence of the polyelectrolytes, but the inhibition was removed by excess of substrate. We propose that the oligomeric state of enzymes causes polyelectrolytes to act on them in a special manner, this special effect differing significantly from the effect of polyelectrolytes on monomeric enzymes. The effect consists in that polyelectrolytes cleave the oligomeric structure of the enzymes, this "cleaving" effect being higher the greater the hydrophobicity of the polyelectrolyte chain.     

The effect of unconscious color hue saturation on emotional state of human

The aim of the study was to investigate influence of color hue saturation on emotional state of human. We use frontal EEG asymmetry to determine subject's emotional state. Our emotional stimuli summon opposite dynamics of frontal EEG asymmetry. Negative stimuli elicits decreasing of the value of frontal EEG asymmetry and positive stimuli increases the value of frontal EEG asymmetry in fronto-polar and frontal leads. Such dynamics of frontal EEG asymmetry point the emotional experience in accordance the stimulus modality. Blue and red color modification of stimuli leads changes in dynamics of frontal EEG asymmetry during presentation of emotional stimuli and after. In fact, that no one subject gave a report about color difference between stimuli during an experiment, we conclude that influence of color modification was unconscious. Our result shows the possibility of unconscious perception color modification to emotional state of human.