Divalent cation binding to ceruloplasmin

Binding of calcium to human and sheep ceruloplasniin was investigated by metal substitution with manganese and competitive displacement of bound manganese by calcium monitored by electron paramagnetic resonance spectroscopy. The K _d for calcium was found to be 1.4mm. Magnesium also bound to ceruloplasmin, with K _d = 0.3 and 0.7 mm for the human and sheep protein, respectively. The thermal stability of ceruloplasmin, as studied by differential scanning calorimetry, was affected by calcium but not by magnesium. A considerable increase of the T _m value, from 73.8 to 83.1°C, was observed for sheep ceruloplasmin in the presence of calcium. The T _m value of the human protein was only slightly altered by calcium (from 85.1 to 87°C). The interaction of ceruloplasmin with the chromatographic material used for its isolation, Sepharose 4B derivatized with chloroethylamine, was weakened by calcium. This allowed us to set up a novel purification scheme that made it possible to efficiently isolate ceruloplasmin and prothrombin from plasma with the same single-step chromatography.     

The ferroportin-ceruloplasmin system and the mammalian iron homeostasis machine: regulatory pathways and the role of lactoferrin

In the last 20 years, several new genes and proteins involved in iron metabolism in eukaryotes, particularly related to pathological states both in animal models and in humans have been identified, and we are now starting to unveil at the molecular level the mechanisms of iron absorption, the regulation of iron transport and the homeostatic balancing processes. In this review, we will briefly outline the general scheme of iron metabolism in humans and then focus our attention on the cellular iron export system formed by the permease ferroportin and the ferroxidase ceruloplasmin. We will finally summarize data on the role of the iron binding protein lactoferrin on the regulation of the ferroportin/ceruloplasmin couple and of other proteins involved in iron homeostasis in inflamed human macrophages.     

Molecular comparison of monocot and dicot U1 and U2 snRNAs

To elucidate differences between the pre-mRNA splicing components in monocots and dicots, we have cloned and characterized several U1 and U2 snRNA sequence variants expressed in wheat seedling nuclei. Primer extension sequencing on wheat and pea snRNA populations has demonstrated that two 5'-terminal nucleotides found in most other U1 snRNAs are missing/modified in many plant U1 snRNAs. Comparison of the wheat U1 and U2 snRNA variants with their counterparts expressed in pea nuclei has defined regions of structural divergence between monocot and dicot U1 and U2 snRNAs. The U1 and U2 snRNA sequences involved in RNA:RNA interaction with pre-mRNAs are absolutely conserved. Significant differences occur between wheat and pea U1 snRNAs in stem I and II structures implicated in the binding of U1-specific proteins suggesting that the monocot and dicot U1-specific snRNP proteins differ in their binding specificities. Stem III structures, which are required in mammalian systems for splicing complex formation but not for U1-specific protein binding, differ more extensively than stems I, II, or IV. In U2 snRNAs, the sequence differences between these two species are primarily localized in stem III and in stem IV which has been implicated in snRNP protein binding. These differences suggest that monocot and dicot U1 and U2 snRNPs represent distinct entities that may have monocot- and dicot-specific snRNP protein variants associated with each snRNA.     

Spin-label and fluorescence labeling studies of the thioester bonds in human alpha 2-macroglobulin

Upon cleavage of the reactive thioester bonds (Cys-949-Glx-952) of tetrameric human alpha 2-macroglobulin (alpha 2M) by methylamine, one sulfhydryl group per alpha 2M subunit is exposed. These identical sulfhydryl group sites were labeled with the thiol-specific nitroxide spin-labels (1-oxy-2,2,5,5-tetramethyl-3-pyrrolin-3-yl)methyl methanethiosulfonate and (1-oxy-2,2,6,6-tetramethyl-4-piperidinyl)methyl methanethiosulfonate, a homologous series of maleimide spin-labels, and the thiol-specific fluorescent probe 2-[(4-maleimidophenyl)amino]naphthalene-6-sulfonic acid sodium salt (MANS). The ESR and fluorescence results showed that these sulfhydryl group sites were at the base of a narrow crevice that is greater than or equal to 8 A deep. Although the bound MANS fluorophore was slightly blue shifted with an enhanced quantum yield vs the free label in water, the environment of the sulfhydryl site appeared to be of a polar nature when compared with the emission maxima in several solvents of varying polarity. The Glx residue participating in the thioester linkage in the intact protein was labeled with 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl. The distance between the Glx and Cys moieties was estimated at greater than or equal to 10-25 A from double spin-labeling experiments.     

Methionine-90-spin-labeled bovine alpha-lactalbumin: electron spin resonance and NMR distance measurements

The unique methionine residue of bovine alpha-lactalbumin was modified by irreversible alkylation with the bromoacetamido nitroxide spin-label 4-(2-bromoacetamido)-2,2,6,6-tetramethylpiperidine-N-oxyl. The line shape of the electron spin resonance (ESR) spectrum was indicative of a fairly mobile spin-label and was sensitive to the calcium-induced conformational change. Paramagnetic broadening of the spin-label ESR lines by a Gd(III) ion substituted at the high-affinity calcium site of the protein yielded a distance between the spin-label and the metal-binding site of 8.0 +/- 1.0 A. The extent of the paramagnetic line broadening by the covalently attached nitroxide spin-label on the proton resonances of several amino acid residues of the protein at 500 MHz allowed estimation of intramolecular distances between the methionine-90 residue and several resolvable protons.     

Intestinal barrier function in response to abundant or depleted mucosal glutathione in Salmonella-infected rats

Background  

Glutathione, the main antioxidant of intestinal epithelial cells, is suggested to play an important role in gut barrier function and prevention of inflammation-related oxidative damage as induced by acute bacterial infection. Most studies on intestinal glutathione focus on oxidative stress reduction without considering functional disease outcome. Our aim was to determine whether depletion or maintenance of intestinal glutathione changes susceptibility of rats to Salmonella infection and associated inflammation.     

Ligands which effect human protein C activation by thrombin

This report documents attempts to mimic the rate enhancement effect of thrombomodulin on human alpha-thrombin-catalyzed activation of human protein C in the absence of exogenous calcium. Specifically the following tryptamine analogs at 1 mM concentration were shown to enhance the protein C activation rate relative to a control with no added effector at pH 8.3 (50 mM Tris-HCl, 0.1 M NaCl, 37 degrees C): serotonin, 1.2; tryptamine, 2.9; 5-fluorotryptamine, 4.4; 6-fluorotryptamine, 7.2. At much higher levels, e.g. 10 mM, all of the above effectors, as well as indole, showed a moderate inhibition of human protein C activation. ATP, a platelet release product, showed a sigmoidal inhibition pattern similar to that found previously for thrombin amidase, clotting, and esterase activity (Conery, B.G., and Berliner, L.J. (1983) Biochemistry 22, 369-375). Overall, the enhancement factors for human alpha-thrombin activation of protein C with the tryptamine analogs described above were remarkable when considering the effect of a simple ligand versus the natural activator, thrombomodulin.     

Cupryphans,metal‐binding,redox‐active,redesigned conopeptides

Contryphans are bioactive peptides, isolated from the venom of marine snails of the genus Conus, which are characterized by the short length of the polypeptide chain and the high degree of unusual post‐translational modifications. The cyclization of the polypeptide chain through a single disulphide bond, the presence of two conserved Pro residues, and the epimerization of a Trp/Leu residue confer to Contryphans a stable and well‐defined structure in solution, conserved in all members of the family, and tolerant to multiple substitutions. The potential of Contryphans as scaffolds for the design of redox‐active (macro)molecules was tested by engineering a copper‐binding site on two different variants of the natural peptide Contryphan‐Vn. The binding site was designed by computational modeling, and the redesigned peptides were synthesized and characterized by optical, fluorescence, electron spin resonance, and nuclear magnetic resonance spectroscopy. The novel peptides, named Cupryphan and Arg–Cupryphan, bind Cu²⁺ ions with a 1:1 stoichiometry and a K_d in the 100 nM range. Other divalent metals (e.g., Zn²⁺ and Mg²⁺) are bound with much lower affinity. In addition, Cupryphans catalyze the dismutation of superoxide anions with an activity comparable to other nonpeptidic superoxide dismutase mimics. We conclude that the Contryphan motif represents a natural robust scaffold which can be engineered to perform different functions, providing additional means for the design of catalytically active mini metalloproteins.