Calcium Sites Research Articles

Structural characteristics of protein binding sites for calcium and lanthanide ions.

Surveys of X-ray structures of Ca2+-containing and lanthanide ion-containing proteins and coordination complexes have been performed and structural features of the metal binding sites compared. A total of 515 structures of Ca2+-containing proteins were considered, although the final data set contained only 44 structures and 60 Ca2+ binding sites with a total of 323 ligands. Eighteen protein structures containing lanthanide ions were considered with a final data set containing eight structures and 11 metal binding sites. Structural features analysed include coordination numbers of the metal ions, the identity of their ligands, the denticity of carboxylate ligands, and the type of secondary structure from which the ligands are derived. Three general types of calcium binding site were identified in the final data set: class I sites supply the Ca2+ ligands from a continuous short sequence of amino acids; class II sites have one ligand supplied by a part of the amino acid sequence far removed from the main binding sequence; and class III sites are created by amino acids remote from one another in the sequence. The abundant EF-hand type of Ca2+ binding site was under-represented in the data set of structures analysed as far as its biological distribution is concerned, but was adequately represented for the chemical survey undertaken. A turn or loop structure was found to provide the bulk of the ligands to Ca2+, but helix and sheet secondary structures are slightly better providers of bidentate carboxylate ligation than turn or loop structures. The average coordination number for Ca2+ was 6.0, though for EF-hand sites it is 7. The average coordination number of a lanthanide ion in an intrinsic protein Ca2+ site was 7.2, but for the adventitious sites was only 4.4. A survey of the Cambridge Structural Database showed there are small-molecule lanthanide complexes with low coordination numbers but it is likely that water molecules, which do not appear in the electron density maps, are present for some lanthanide sites in proteins. A detailed comparison of the well-defined Ca2+ and lanthanide ion binding sites suggests that a reduction of hydrogen bonding associated with the ligating residues of the binding sites containing lanthanide ions may be a response to the additional positive charge of the lanthanide ion. Major structural differences between Ca2+ binding sites with weak and strong binding affinities were not obvious, a consequence of long-range electrostatic interactions and metal ion-induced protein conformational changes modulating affinities.

Density-functional theory calculations of the interaction of protons and water with low-coordinated surface sites of calcium oxide

Electronic structure calculations, using the density-functional theory within the generalized-gradient approximation and ultrasoft pseudopotentials, have been used to investigate the interaction of protons and water with five-, four-, and three-coordinated surface sites of calcium oxide, modeled by the {100}, {310}, and microfacetted {111} surface, respectively. The calculated structural parameters of bulk CaO are found to be in good agreement with experiment. The nonhydrated {100} surface shows negligible ionic relaxation from bulk termination due to the minimal distortion of the electron density in the surface layer and electron-density plots show the crystal to be strongly ionic. On the microfacetted {111} surface redistribution of the electron density along the bond between three-coordinated calcium ion and four-coordinated oxygen ions leads to the contraction of the bond lengths and relaxation of the surface calcium into the surface. Absorption of water at the six-coordinated ions in the bulk crystal is calculated to be highly endothermic. On the five-coordinated {100} surface sites, water molecules, which were initially dissociatively adsorbed, recombine to form associatively adsorbed species with an adsorption energy of approximately 65 ${\mathrm{kJmol}}^{\mathrm{\ensuremath{-}}1}$, indicating physisorption. The water molecules are adsorbed by their oxygen ion to surface calcium ions but electron-density plots show additional interactions between surface anions and hydrogen atoms. On four- and three-coordinated calcium sites, adsorbed water molecules dissociate to form hydroxyl groups indicating the higher reactivity of the lower-coordinated species. The adsorption energy at the four-coordinated calcium site is higher (156 ${\mathrm{kJmol}}^{\mathrm{\ensuremath{-}}1}$) than at the three-coordinated site (127 ${\mathrm{kJmol}}^{\mathrm{\ensuremath{-}}1}$), due to bonding of the OH group to two surface calcium ions. When calcium ions are replaced by two protons each we find that the replacement energy decreases by approximately 70 ${\mathrm{kJmol}}^{\mathrm{\ensuremath{-}}1}$ per loss of bond, from +21 ${\mathrm{kJmol}}^{\mathrm{\ensuremath{-}}1}$ for the six-coordinated calcium ions in the bulk to -187 ${\mathrm{kJmol}}^{\mathrm{\ensuremath{-}}1}$ for the three-coordinated calcium ions on the faceted {111} surface.

Effect of doping Ba, Y V, Zn and Sn on BSCCO superconducting ceramics

The effect of doping on the superconductivity in Bi/sub 2/Pb/sub 0.6/Sr/sub 2/Ca/sub 2-x/R/sub x/Cu/sub 3/O/sub /spl delta// (R=Ba,Y,V,Zn,Sn) has been investigated by resistivity, ac susceptibility measurements and x-ray diffraction (XRD) analysis. All the dopants were incorporated in the calcium site with x ranging from 0.00 to 0.10. The temperature dependence of electrical resistance and ac susceptibility measurements was studied for these samples. The nature of the temperature dependence of the resistance curves indicates the presence of a superconducting transition between grains coupled by weak links. From the XRD data, it is observed that the volume percentage of the 2223 phase decreases with the increasing doping concentration. The presence of low T/sub c/ phase is also visible in the real component (/spl chi/'), of the susceptibility data, while the imaginary component (/spl chi/), shows a shift towards lower temperature in the intergranular coupling peak, T/sub p/, as the dopant concentration increases. All the doping elements do not enhance the T/sub c/ zero but decreases its value from /spl sim/104 K to 88 K, 53 K, 79.6 K, 58 K and 53 K for Ba, Y, V, Zn and Y respectively for x=0.10. Barium doped samples are least affected by compositional variation and the zero resistance values are higher than that of the other doped samples.

VARIATIONS IN CALCIUM USE BY BIRDS DURING THE BREEDING SEASON

Abstract During the breeding season birds need increased amounts of dietary calcium during egg laying and nestling rearing. Passerines acquire calcium shortly before and during laying and do not store calcium for egg formation. Many passerines need to eat extraneous calcium in the form of snail shells or calcareous grit to acquire the element. However, very little is known about variation in the dependency of birds on extraneous calcium, and where this calcium is acquired. Using reports from volunteers who provided extraneous calcium sources at feeder sites across North America we documented that (1) the use of extraneous calcium varied during the breeding season; (2) the location in which species preferentially took the calcium varied with typical feeding location, aside from swallows which, somewhat surprisingly, favored calcium offered on the ground; (3) Brown-headed Cowbirds (Molothrus ater) did not visit calcium sites more often than other blackbirds; (4) calcium use varied among species with thrushe...

Structure and function of laminin LG modules.

Laminin G domain-like (LG) modules of approximately 180-200 residues are found in a number of extracellular and receptor proteins and often are present in tandem arrays. LG modules are implicated in interactions with cellular receptors (integrins, alpha-dystroglycan), sulfated carbohydrates and other extracellular ligands. The recently determined crystal structures of LG modules of the laminin alpha2 chain reveal a compact beta sandwich fold and identify a novel calcium binding site. Binding epitopes for heparin, sulfatides and alpha-dystroglycan have been mapped by site-directed mutagenesis and show considerable overlap. The epitopes are located in surface loops around the calcium site, which in other proteins (agrin, neurexins) are modified by alternative splicing. Efficient ligand binding often requires LG modules to be present in tandem. The close proximity of the N- and C-termini in the LG module, as well as a unique link region between laminin LG3 and LG4, impose certain constraints on the arrangement of LG tandems. Further modifications may be introduced by proteolytic processing of laminin G domains, which is known to occur in the alpha2, alpha3 and alpha4 chains.

Evidence for Gelsolin as a Corneal Crystallin in Zebrafish

We have shown that gelsolin is one of the most prevalent water-soluble proteins in the transparent cornea of zebrafish. There are also significant amounts of actin. In contrast to actin, gelsolin is barely detectable in other eye tissues (iris, lens, and remaining eye) of the zebrafish. Gelsolin cDNA hybridized intensely in Northern blots to RNA from the cornea but not from the lens, brain, or headless body. The deduced zebrafish gelsolin is approximately 60% identical to mammalian cytosolic gelsolin and has the characteristic six segmental repeats as well as the binding sites for actin, calcium, and phosphatidylinositides. In situ hybridization tests showed that gelsolin mRNA is concentrated in the zebrafish corneal epithelium. The zebrafish corneal epithelium stains very weakly with rhodamine-phalloidin, indicating little F-actin in the cytoplasm. In contrast, the mouse corneal epithelium contains relatively little gelsolin and stains intensely with rhodamine-phalloidin, as does the zebrafish extraocular muscle. We propose, by analogy with the diverse crystallins of the eye lens and with the putative enzyme-crystallins (aldehyde dehydrogenase class 3 and other enzymes) of the mammalian cornea, that gelsolin and actin-gelsolin complexes act as water-soluble crystallins in the zebrafish cornea and contribute to its optical properties.

Structural analysis of a chimeric bacterial alpha-amylase. High-resolution analysis of native and ligand complexes.

Several chimeric alpha-amylases genes were constructed by an in vivo recombination technique from the Bacillus amyloliquefaciens and Bacillus licheniformis genes. One of the fusion amylases (hereafter BA2), consisting of residues 1-300 from B. amyloliquefaciens and 301-483 from B. licheniformis, has been extensively studied by X-ray crystallography at resolutions between 2.2 and 1.7 A. The 3-dimensional structure of the native enzyme was solved by multiple isomorphous replacement, and refined at a resolution of 1.7 A. It consists of 483 amino acids, organized similarly to the known B. lichiniformis alpha-amylase structure [Machius et al. (1995) J. Mol. Biol. 246, 545-559], but features 4 bound calcium ions. Two of these form part of a linear cluster of three ions, the central ion being attributed to sodium. This cluster lies at the junction of the A and B domains with one calcium of the cluster structurally equivalent to the major Ca(2+) binding site of fungal alpha-amylases. The third calcium ion is found at the interface of the A and C domains. BA2 contains a fourth calcium site, not observed in the B. licheniformis alpha-amylase structure. It is found on the C domain where it bridges the two beta-sheets. Three acid residues (Glu261, Asp328, and Asp231) form an active site similar to that seen in other amylases. In the presence of TRIS buffer, a single molecule of TRIS occupies the -1 subsite of the enzyme where it is coordinated by the three active-center carboxylates. Kinetic data reveal that BA2 displays properties intermediate to those of its parents. Data for crystals soaked in maltooligosaccharides reveal the presence of a maltotriose binding site on the N-terminal face of the (beta/alpha)(8) barrel of the molecule, not previously described for any alpha-amylase structure, the biological function of which is unclear. Data for a complex soaked with the tetrasaccharide inhibitor acarbose, at 1.9 A, reveal a decasaccharide moiety, spanning the -7 to +3 subsites of the enzyme. The unambiguous presence of three unsaturated rings in the (2)H(3) half-chair/(2)E envelope conformation, adjacent to three 6-deoxypyranose units, clearly demonstrates synthesis of this acarbose-derived decasaccharide by a two-step transglycosylation mechanism.

Structure of a C-type Carbohydrate Recognition Domain from the Macrophage Mannose Receptor

The mannose receptor of macrophages and liver endothelium mediates clearance of pathogenic organisms and potentially harmful glycoconjugates. The extracellular portion of the receptor includes eight C-type carbohydrate recognition domains (CRDs), of which one, CRD-4, shows detectable binding to monosaccharide ligands. We have determined the crystal structure of CRD-4. Although the basic C-type lectin fold is preserved, a loop extends away from the core of the domain to form a domain-swapped dimer in the crystal. Of the two Ca(2+) sites, only the principal site known to mediate carbohydrate binding in other C-type lectins is occupied. This site is altered in a way that makes sugar binding impossible in the mode observed in other C-type lectins. The structure is likely to represent an endosomal form of the domain formed when Ca(2+) is lost from the auxiliary calcium site. The structure suggests a mechanism for endosomal ligand release in which the auxiliary calcium site serves as a pH sensor. Acid pH-induced removal of this Ca(2+) results in conformational rearrangements of the receptor, rendering it unable to bind carbohydrate ligands.

Effects of an anticariogenic casein phosphopeptide on calcium diffusion in streptococcal model dental plaques

Casein phosphopeptides (CPP) stabilize amorphous calcium phosphate (ACP) and may be used to localize ACP in dental plaque, maintaining a state of supersaturation with respect to tooth enamel, reducing demineralization and enhancing remineralization. The aim here was to investigate these effects by measuring the effect of CPP–ACP on calcium diffusion in plaque. Using Dibdin’s effusion system, calcium diffusion was measured in streptococcal model plaques. This demonstrated that by providing a large number of possible binding sites for calcium, 0.1% CPP–ACP reduces the calcium diffusion coefficient by about 65% at pH 7 and 35% at pH 5. Hence, CPP–ACP binds well to plaque, providing a large calcium reservoir within the plaque and slowing diffusion of free calcium. This is likely to restrict mineral loss during a cariogenic episode and provide a potential source of calcium for subsequent remineralization. Overall, once in place, CPP–ACP will restrict the caries process.

X-Ray Absorption Spectroscopy of Strontium(II) Coordination: I. Static and Thermal Disorder in Crystalline, Hydrated, and Precipitated Solids and in Aqueous Solution

Detailed analyses of crystalline, hydrated, and precipitated strontium compounds and an aqueous strontium solution by synchrotron extended X-ray absorption fine structure (EXAFS) were used to quantify local thermal and static disorder and to characterize strontium coordination in a variety of oxygen-ligated bonding environments. Analysis of anharmonic vibrational disorder (i.e., significant contribution from a third cumulant term (C3) in the EXAFS phase-shift function) in compounds with low and high static disorder around strontium showed that first-shell anharmonic contributions were generally not significant above experimental error in the EXAFS fits (R±0.02 Å with and without C3). The only case in which a significant apparent decrease in Sr–O distance was observed with increasing temperature, and for which a third cumulant term was significant, was for dilute strontium in aqueous solution. Empirical parameterization of Debye–Waller factor (σ2) for strontium compounds as a function of backscatterer atomic number (Z), interatomic Sr–Z distance, and temperature of spectral data collection showed systematic increases in σ2 as a function of increasing temperature and Sr–Z bond length. At values of σ2 greater than ≈0.025 Å2 (for N<12 and RSr-Z>3 Å), backscattering was generally not significant above noise levels in spectra of compounds of known crystal structure. Comparison of the EXAFS spectra of freshly precipitated SrCO3 (spectra collected wet) to that of dry, powdered strontianite (SrCO3(s)) indicated no significant differences in the local atomic structure around strontium. Analysis of partially hydrated strontium in natural Ca-zeolite (heulandite) showed that strontium is substituted only in the calcium (Ca2) site. Backscattering from aluminum and silicon atoms in the zeolite framework were apparent in the EXAFS spectra at low and room temperature at distances from central strontium of <4.2 Å. Comparison of strontium structural coordination determined in this and previous studies suggests that previous EXAFS determinations of hydrated strontium may have underestimated first-shell interatomic distances and coordination numbers because minor contributions to the EXAFS phase-shift and amplitude functions were not accounted for, either theoretically or empirically.

NMR Solution Structure of Complement-like Repeat CR3 from the Low Density Lipoprotein Receptor-related Protein: EVIDENCE FOR SPECIFIC BINDING TO THE RECEPTOR BINDING DOMAIN OF HUMAN α2-MACROGLOBULIN

We have used NMR methods to determine the structure of the calcium complex of complement-like repeat 3 (CR3) from the low density lipoprotein receptor-related protein (LRP) and to examine its specific interaction with the receptor binding domain of human alpha(2)-macroglobulin. CR3 is one of eight related repeats that constitute a major ligand binding region of LRP. The structure is very similar in overall fold to homologous complement-like repeat CR8 from LRP and complement-like repeats LB1, LB2, and LB5 from the low density lipoprotein receptor and contains a short two-strand antiparallel beta-sheet, a one turn alpha-helix, and a high affinity calcium site with coordination from four carboxyls and two backbone carbonyls. The surface electrostatics and topography are, however, quite distinct from each of these other repeats. Two-dimensional (1)H,(15)N-heteronuclear single quantum coherence spectra provide evidence for a specific, though relatively weak (K(d) approximately 140 microM), interaction between CR3 and human alpha2-macroglobulin receptor binding domain that involves a contiguous patch of surface residues in the central region of CR3. This specific interaction is consistent with a mode of LRP binding to ligands that uses contributions from more than one domain to generate a wide array of different binding sites, each with overall high affinity.

Hemophilia B with Mutations at Glycine-48 of Factor IX Exhibited Delayed Activation by the Factor VIIa-tissue Factor Complex

Gly-48 is in the conserved DGDQC sequence (residues 47-51 of human factor IX) of the first EGF (EGF-1)-like domain of factor IX. The importance of the Gly-48 is manifested by two hemophilia B patients; factor IXTainan and factor IXMalmo27, with Gly-48 replaced by arginine (designated IXG48R) and valine (IXG48V), respectively. Both patients were CRM+ exhibiting mild hemophilic episodes with 25% (former) and 19% (latter) normal clotting activities. We characterize both factor IX variants to show the roles of Gly-48 and the conservation of the DGDQC sequence in factor IX. Purified plasma and recombinant factor IX variants exhibited approximately 26%-27% normal factor IX's clotting activities with G48R or G48V mutation. Both variants depicted normal quenching of the intrinsic fluorescence by increasing concentrations of calcium ions and Tb3+, indicating that arginine and valine substitution for Gly-48 did not perturb the calcium site in the EGF-1 domain. Activation of both mutants by factor XIa appeared normal. The reduced clotting activity of factors IXG48R and IXG48V was attributed to the failure of both mutants to cleavage factor X: in the presence of only phospholipids and calcium ions, both mutants showed a 4 to approximately 7-fold elevation in Km, and by adding factor VIIIa to the system, although factor VIIIa potentiated the activation of factor X by the mutants factor IXaG48R and factor IXaG48V, a 2 to approximately 3-fold decrease in the catalytic function was observed with the mutant factor IXa's, despite that they bound factor VIIIa on the phospholipid vesicles with only slightly reduced affinity when compared to wild-type factor IXa. The apparent Kd for factor VIIIa binding was 0.83 nM for normal factor IXa, 1.74 nM for IXaG48R and 1.4 nM for IXaG48V. Strikingly, when interaction with the factor VIIa-TF complex was examined, both mutations were barely activated by the VIIa-TF complex and they also showed abnormal interaction with VIIa-TF in bovine thromboplastin-based PT assays. Taken together, our results suggest that mutations at Gly-48 altered the interaction of factor IX with its extrinsic pathway activator (VIIa-TF complex), its macromolecular substrate (factor X), and its cofactor (factor VIIIa).

The partitioning of arsenic during pulverized coal combustion

An important issue facing the coal-fired electric utility industry is the environmental impact resulting from the release of arsenic contained in the coal. Depending on the forms of occurence in the coal and transformation mechanism involved, arsenic-containing compounds may exit the furnace in the vapor, as part of submicron size aerosol particles, or as part of larger, supermicron particles. The specific path by which arsenic transforms from the vapor phase to the condensed phase is important in assessing the toxicity of the waste product. This paper documents research exploring the partitioning of arsenic during the combustion of five well-characterized pulverized coals under commercially relevant conditions in a welldescribed laboratory combustion environment. Insight is gained into the timing of partitioning because this combustions system allows comparison of furnace vapor and particulate at different locations in the furnace. The partitioning of arsenic is governed primarily by the extent of arsenic volatilization during combustion. Volatilized arsenic will heterogeneously transform to both submicron and supermicron particles primarily by reaction with active cation sites. For many coals, arsenic appears to be highly reactive with active calcium sites to form calcium arsenates. The transformation process is typically dominated by surface reactivity rather than by gas film diffusion. Due to the similarities in the solubility characteristics of As 2 O 3 and calcium arsenate, leaching studies provide little insight into the form of arsenic present on fly ash surfaces.

Structural and superconducting properties of (Hg0.7Bi0.3)Sr2Can-1CunOx (n = 3,4) superconductors synthesized under high pressure

The third and fourth members (n = 3 and 4) of Hg-Sr, HgSr2Can-1CunOx, series have been stabilized by Bi under high pressure. The phase formation of these phases is found to be sensitive to the nominal oxygen content, x, in the starting mixture. High phase purity is obtained with nominal oxygen contents of 8.55 and 10.15 for n = 3 and 4, respectively. The combined XRD and EDS analyses of the phase with n = 3 show the presence of an excess of strontium in the calcium sites. The sample with nominal composition (Hg0.7Bi0.3)Sr2Ca2Cu3O8.55 was overdoped and exhibited bulk superconductivity below 105 and 116 K in the as-prepared form and after annealing in Ar/H2 atmosphere, respectively. On the other hand, the highest Tc of the sample with nominal composition (Hg0.7Bi0.3)Sr2Ca3Cu4O10.15 was 96 K in the as-prepared form and did not change with annealing.

EXAFS and XANES Analysis of Plutonium and Cerium Edges From Titanate Ceramics for Fissile Materials Disposal

We report x-ray absorption near edge structure (XANES) and extended x-ray absorption fine structure (EXAFS) spectra from the plutonium L{sub III} edge and XANES from the cerium L{sub II} edge in prototype titanate ceramic hosts. The titanate ceramics studied are based upon the hafnium-pyrochlore and zirconolite mineral structures and will serve as an immobilization host for surplus fissile materials, containing as much as 10 weight % fissile plutonium and 20 weight % (natural or depleted) uranium. Three ceramic formulations were studied: one employed cerium as a ''surrogate'' element, replacing both plutonium and uranium in the ceramic matrix, another formulation contained plutonium in a ''baseline'' ceramic formulation, and a third contained plutonium in a formulation representing a high-impurity plutonium stream. The cerium XANES from the surrogate ceramic clearly indicates a mixed III-IV oxidation state for the cerium. In contrast, XANES analysis of the two plutonium-bearing ceramics shows that the plutonium is present almost entirely as Pu(IV) and occupies the calcium site in the zirconolite and pyrochlore phases. The plutonium EXAFS real-space structure shows a strong second-shell peak, clearly distinct from that of PuO{sub 2}, with remarkably little difference in the plutonium crystal chemistry indicated between the baseline and high-impurity formulations.

The nucleotide-binding site of the sarcoplasmic reticulum Ca-ATPase is conformationally altered in aged skeletal muscle.

Cellular conditions in senescent skeletal muscle have been shown to result in the loss of conformational stability of the sarcoplasmic reticulum (SR) Ca-ATPase. To identify underlying structural features of age-modified Ca-ATPase, we have utilized the fluorescence properties of protein-bound probes to assess both local and global structure. We find conformational changes that include an age-related decrease in the apparent binding affinity to high affinity calcium sites detected by fluorescence signals in both tryptophans within nearby membrane-spanning helices and fluorescein isothiocyanate (FITC) bound distally to Lys(515) within the nucleotide-binding site. In addition, a substantial (80%) age-related increase in the accessibility to soluble quenchers of fluorescence of FITC is observed without concomitant changes in bimolecular quenching constants (k(q)) for protein-bound IAEDANS, also within the nucleotide-binding domain, and tryptophans within the membrane. Using fluorescence resonance energy transfer to measure distances between IAEDANS and FITC across the nucleotide-binding domain, we find no significant age-related change in the mean donor-acceptor distance; however, significant increases are observed in the conformational heterogeneity of this domain, as assessed by the width at half-maximum (HW) of the distance distribution, increasing with age from 29.4 +/- 0.8 A to 42.5 +/- 1. 1 A. Circular dichroism indicates that the average secondary structure is unaltered with age. Thus, these data suggest tertiary structural alterations in specific regions around the nucleotide-binding site rather than global conformational changes.

Role of metal ions in the T- to R-allosteric transition in the insulin hexamer.

The role of metal ions in the T- to R-allosteric transition is ascertained from the investigation of the T- to R-allosteric transition of transition metal ions substituted-insulin hexamers, as well as from the kinetics of their dissociation. These studies establish that ligand field stabilization energy (LFSE), coordination geometry preference, and the Lewis acidity of the metal ion in the zinc sites modulate the T- to R-state transition. (1)H NMR, (113)Cd NMR, and UV-vis measurements demonstrate that, under suitable conditions, Fe2+/3+, Ni2+, and Cd2+ bind insulin to form stable hexamers, which are allosteric species. (1)H NMR R-state signatures are elicited by addition of phenol alone in the case of Ni(II)- and Cd(II)-substituted insulin hexamers. The Fe(II)-substituted insulin hexamer is converted to the ferric analogue upon addition of phenol. For the Fe(III)-substituted insulin hexamer, appearance of (1)H NMR R-state signatures requires, additionally to phenol, ligands containing a nitrogen that can donate a lone pair of electrons. This is consistent with stabilization of the R-state by heterotropic interactions between the phenol-binding pocket and ligand binding to Fe(III) in the zinc site. UV-vis measurements indicate that the (1)H NMR detected changes in the conformation of the Fe(III)-insulin hexamer are accompanied by a change in the electronic structure of the iron site. Kinetic measurements of the dissociation of the hexamers provide evidence for the modulation of the stability of the hexamer by ligand field stabilization effects. These kinetic studies also demonstrate that the T- to R-state transition in the insulin hexamer is governed by coordination geometry preference of the metal ion in the zinc site and the compatibility between Lewis acidity of the metal ion in the zinc site and the Lewis basicity of the exogenous ligands. Evidence for the alteration of the calcium site has been obtained from (113)Cd NMR measurements. This finding adds to the number of known conformational changes that occur during the T- to R-transition and is an important consideration in the formulation of allosteric mechanisms of the insulin hexamer.

Changes in ryanodine receptor-mediated calcium release during skeletal muscle differentiation.

We have observed a disparity between the actions of caffeine and ryanodine, two agents known to affect the same site of intracellular calcium (Ca2+) release in muscle. The site of intracellular Ca2+ release, the ryanodine receptor (RyR), is established as the route of Ca2+ movement from the sarcoplasmic reticulum (SR) to the cytosol during excitation-contraction coupling. We measured Ca2+ release fluorimetrically in both saponin-permeabilized and intact L6 cells, in response to known modulators (i.e., caffeine and ryanodine), during differentiation in vitro. The undifferentiated L6 cells showed little response to caffeine. However, a substantial caffeine-induced calcium release (caffCR) was evident by Day 3 of differentiation, and was nearly maximal by Day 7 of differentiation. By contrast, ryanodine failed to stimulate Ca2+ release until Day 4, lagging behind the caffeine response. Ryanodine-stimulated Ca2+ release was also maximal by Day 7. Higher concentrations of ryanodine, known to inhibit Ca2+ release, only began to affect caffCR at Day 4, indicating that cells were insensitive to both ryanodine stimulation and ryanodine inhibition prior to this time. Most of the results could be obtained both in permeabilized and intact cells. Using intact cells, we measured the time course of K+ -dependent (i.e., depolarization-induced) Ca2+ release. This time course matched caffeine and not ryanodine-induced Ca2+ release suggesting the action of caffeine was not due to Ca2+ release unrelated to excitation-contraction coupling. These findings suggest that ryanodine binding sites on the RyR may not be functional at early stages of muscle development, that ryanodine sensitivity is a poor indicator of Ca2+ flux through the RyR, or that other proteins are involved in Ca2+ release under certain circumstances.

Changes in Ryanodine Receptor-Mediated Calcium Release During Skeletal Muscle Differentiation

We have observed a disparity between the actions of caffeine and ryanodine, two agents known to affect the same site of intracellular calcium (Ca2+) release in muscle. The site of intracellular Ca2+ release, the ryanodine receptor (RyR), is established as the route of Ca2+ movement from the sarcoplasmic reticulum (SR) to the cytosol during excitation-contraction coupling. We measured Ca2+ release fluorimetri-cally in both saponin-permeabilized and intact L6 cells, in response to known modulators (i.e., caffeine and ryanodine), during differentiation in vitro. The undifferentiated L6 cells showed little response to caffeine. However, a substantial caffeine-induced calcium release (caffCR) was evident by Day 3 of differentiation, and was nearly maximal by Day 7 of differentiation. By contrast, ryanodine failed to stimulate Ca2+ release until Day 4, lagging behind the caffeine response. Ryanodine-stimulated Ca2+ release was also maximal by Day 7. Higher concentrations of ryanodine, known to inhibit Ca2+ release, only began to affect caffCR at Day 4, indicating that cells were insensitive to both ryanodine stimulation and ryanodine inhibition prior to this time. Most of the results could be obtained both in permeabilized and intact cells. Using intact cells, we measured the time course of K+-dependent (i.e., depolarization-induced) Ca2+ release. This time course matched caffeine and not ryanodine-induced Ca2+ release suggesting the action of caffeine was not due to Ca2+ release unrelated to excitation-contraction coupling. These findings suggest that ryanodine binding sites on the RyR may not be functional at early stages of muscle development, that ryanodine sensitivity is a poor indicator of Ca2+ flux through the RyR, or that other proteins are involved in Ca2+ release under certain circumstances.

Adsorption of glycosaminoglycans onto coral--a new possible implant biomaterials for regeneration therapy.

The adsorption of glycosaminoglycans (heparin, heparan sulfate, dermatan sulfate, highly sulfated chondroitin sulfate, chondroitin sulfate, and hyaluronan) onto coral has been investigated. Granules of natural coral of specific diameter, between 100 and 500 microm, having high content of calcium (> 98%) and a homogeneous surface adsorb glycosaminoglycans with different capacity. Heparin (maximum adsorption 1.29 +/- 0.10 mg/20 mg of coral, 6.45% w/w) is adsorbed more than highly sulfated chondroitin sulfate species (maximum adsorption of 0.90 +/- 0.06 mg/20 mg of coral, 4.50% w/w), chondroitin sulfate (maximum adsorption of 0.72 +/- 0.06 mg/20 mg of coral, 3.60% w/w), dermatan sulfate (maximum adsorption of 0.70 +/- 0.06 mg/20 mg of coral, 3.50% w/w) and heparan sulfate (maximum adsorption of 0.72 +/- 0.07 mg/20 mg of coral, 3.60% w/w). Hyaluronan is not adsorbed onto granules of coral. The percentage adsorption of polyanions onto coral depends mainly on their charge density, with sulfate groups being more important than carboxyl groups. This study found no evidence that iduronic acid is more important than glucuronic acid and no role of molecular mass on the adsorption of polysaccharides onto coral was found. The adsorption of glycosaminoglycans is driven by electrostatic interactions with calcium sites of coral that are dependent on pH and blocked in the presence of large amounts of salt. Due to these peculiar properties, the combination of granules of natural coral with glycosaminoglycans makes this material potentially useful in osseointegration in bone metabolism or periodontal therapy.