Characterization of the Single Tyrosine Containing Troponin C from Lungfish White Muscle. Comparison with Several Fast Skeletal Muscle Troponin C's from Fish Species

Troponin C molecules from fast skeletal muscle of the following fish species (trout, whiting, lungfish, tilapia, and cod) have been purified to homogeneity. Upon binding of Ca²⁺ or Mg²⁺, lungfish troponin C is the only troponin C from fish white muscle to show the typical increase of tyrosine fluorescence emission quantum yield reported for rabbit fast skeletal muscle troponin C. The increase of tyrosine fluorescence signal occurring upon Ca²⁺ and Mg²⁺ titration of lungfish troponin C has been used to determine the corresponding affinity constants. With K_(Ca) = 7.0 10⁷ M⁻¹ and K_(Mg) = 3.6 10³ M⁻¹, the sites probed by the tyrosine residue of lungfish troponin C are typical of the COOH-terminal domain of fast skeletal troponin C's. The amino acid sequencing of the tyrosine containing tryptic peptides has allowed us to position the single tyrosine residue at position 7 in the Ca²⁺ binding loop of the third site, in identical position to Tyr109 of troponin C from rabbit fast skeletal muscle. Metal ion binding studies followed by intrinsic fluorescence or Tb³⁺ luminescence indicate that the conformation of the structural domain of lungfish troponin C with one metal ion bound is close to the physiological conformation of this domain.     

Terbium binding to axonal membrane vesicles from lobster (Homarus Americanus) peripheral nerve. A probe of calcium binding sites

Tb³⁺, a fluorescent trivalent cation with physicochemical properties similar to Ca²⁺, binds to peripheral nerve membrane vesicles prepared from the walking leg nerve bundle of the lobster (Homarus americanus). Saturable binding is measured for at least two classes of binding site. Bound Tb³⁺ can be displaced by other cations in the order: Ca²⁺ > Mg²⁺ = Zn²⁺ > NH₄⁺. The binding of Tb³⁺ to the lower affinity site (K_D(app) = 6.0 μM) is inhibitable by Na⁺, Mg²⁺ and Ca²⁺, whereas the higher affinity site (K_D(app) = 2.2 μM) is only sensitive to Ca²⁺. Using this spectral probe the role of Ca²⁺ in peripheral nerve membrane function can be investigated.     

Binding and aggregation of human μ-calpain by terbium ion

Human μ-calpain is activated maximally by 100–200 μM Ca²⁺. Both the 80 kDa and 29 kDa subunits of μ-calpain have a EF-hand type calcium-binding domain. It is known that trivalent terbium ion (Tb³⁺) mimics Ca²⁺ in many biological systems. We found that Tb³⁺ alone transiently activated calpain. However, in the presence of Ca²⁺, Tb³⁺ inhibited μ-calpain with an IC₅₀ of about 100 μM. As high as 10 mM Ca²⁺ did not significantly shift the IC₅₀ of Tb³⁺. Preincubating μ-calpain by Ca²⁺ (before Tb³⁺ and substrate were added) did not diminish the inhibition by Tb³⁺. On the other hand, pretreating μ-calpain with Tb³⁺ produced that Tb³⁺ has a slow dissociation rate for the calcium-binding sites when compared to Ca²⁺. Electrophoretic analysis revealed that terbium ion transiently activated μ-calpain followed by the aggregation of the proteinase.     

Kinetic Mechanism of Ca2+-controlled Changes of Skeletal Troponin I in Psoas Myofibrils

Conformational changes in the skeletal troponin complex (sTn) induced by rapidly increasing or decreasing the [Ca²⁺] were probed by 5-iodoacetamidofluorescein covalently bound to Cys-133 of skeletal troponin I (sTnI). Kinetics of conformational changes was determined for the isolated complex and after incorporating the complex into rabbit psoas myofibrils. Isolated and incorporated sTn exhibited biphasic Ca²⁺-activation kinetics. Whereas the fast phase (k_obs∼1000 s⁻¹) is only observed in this study, where kinetics were induced by Ca²⁺, the slower phase resembles the monophasic kinetics of sTnI switching observed in another study (Brenner and Chalovich. 1999. Biophys. J. 77:2692–2708) that investigated the sTnI switching induced by releasing the feedback of force-generating cross-bridges on thin filament activation. Therefore, the slower conformational change likely reflects the sTnI switch that regulates force development. Modeling reveals that the fast conformational change can occur after the first Ca²⁺ ion binds to skeletal troponin C (sTnC), whereas the slower change requires Ca²⁺ binding to both regulatory sites of sTnC. Incorporating sTn into myofibrils increased the off-rate and lowered the Ca²⁺ sensitivity of sTnI switching. Comparison of switch-off kinetics with myofibril force relaxation kinetics measured in a mechanical setup indicates that sTnI switching might limit the rate of fast skeletal muscle relaxation.     

Significance of Troponin Dynamics for Ca2+-mediated Regulation of Contraction and Inherited Cardiomyopathy

Ca²⁺ dissociation from troponin causes cessation of muscle contraction by incompletely understood structural mechanisms. To investigate this process, regulatory site Ca²⁺ binding in the NH₂-lobe of subunit troponin C (TnC) was abolished by mutagenesis, and effects on cardiac troponin dynamics were mapped by hydrogen-deuterium exchange (HDX)-MS. The findings demonstrate the interrelationships among troponin''s detailed dynamics, troponin''s regulatory actions, and the pathogenesis of cardiomyopathy linked to troponin mutations. Ca²⁺ slowed HDX up to 2 orders of magnitude within the NH₂-lobe and the NH₂-lobe-associated TnI switch helix, implying that Ca²⁺ greatly stabilizes this troponin regulatory region. HDX of the TnI COOH terminus indicated that its known role in regulation involves a partially folded rather than unfolded structure in the absence of Ca²⁺ and actin. Ca²⁺-triggered stabilization extended beyond the known direct regulatory regions: to the start of the nearby TnI helix 1 and to the COOH terminus of the TnT-TnI coiled-coil. Ca²⁺ destabilized rather than stabilized specific TnI segments within the coiled-coil and destabilized a region not previously implicated in Ca²⁺-mediated regulation: the coiled-coil''s NH₂-terminal base plus the preceding TnI loop with which the base interacts. Cardiomyopathy-linked mutations clustered almost entirely within influentially dynamic regions of troponin, and many sites were Ca²⁺-sensitive. Overall, the findings demonstrate highly selective effects of regulatory site Ca²⁺, including opposite changes in protein dynamics at opposite ends of the troponin core domain. Ca²⁺ release triggers an intramolecular switching mechanism that propagates extensively within the extended troponin structure, suggests specific movements of the TnI inhibitory regions, and prominently involves troponin''s dynamic features.     

Proton relaxation rate and fluorometric studies of manganese and rare earth binding to concanavalin A

The binding of Mn²⁺, Ca²⁺, and rare earth ions to apoconcanavalin A has been studied by water proton relaxation enhancement, electron paramagnetic resonance spectroscopy, and fluorescence spectroscopy. An electron paramagnetic resonance and water proton relaxation rate study of the titration of apoconcanavalin A with Mn²⁺ gives evidence of two equivalent binding sites per monomer with K_D = 50 μm ± 4 μm. When a similar Mn²⁺ titration of apoconcanavalin A is performed in the presence of Ca²⁺ ion, very little free Mn²⁺ is detected by electron paramagnetic resonance until the two Mn²⁺ binding sites per monomer are filled. The substitution of a rare earth ion for Ca²⁺ ion in the above experiment often resulted in a slight displacement of Mn²⁺ from the transition metal site as detected by electron paramagnetic resonance. A water proton relaxation rate study of the titration of apoconcanavalin A with Gd³⁺ reflects two binding sites with a K_D = 40 μm ± 4 μm and two with a K_D = 200 μm ± 50 μm. The fluorescence emission spectrum of concanavalin A (λ_em = 340 nm) is slightly quenched by the addition of Tb³⁺ while Tb³⁺ fluorescence is greatly enhanced. A fluorometric titration of apoconcanavalin A with Tb³⁺ also reflects two sites with a K_D = 40 μm ± 15 μm and two with a K_D = 270 μm ± 50 μm.     

Cytotoxic Effect of Photoluminescent RE3+ Doped Ca3(PO4)2 Nanorods on Breast Cancer Cell Lines

BackgroundBreast cancer reported in the young women exhibits high local and distant recurrence and a poor prognosis. Rare earth doped calcium phosphate phosphors have been extensively investigated due to their unique applications in biomedicine.MethodsIn the current study, Tb³⁺, Ce³⁺ doped Ca₃(PO₄)₂ phosphor were prepared by hydrothermal method at 150 °C using citric acid as additive and characterized by PXRD, FT-IR, TG-DTA, EDX, TEM and PL techniques. The photoluminescence properties of Tb³⁺, Ce³⁺ doped Ca₃(PO₄)₂ phosphor was investigated upon photo excitation at 240 nm. Antiproliferative activity was evaluated by MTT, BrdU proliferation, ELISA, Methylene blue and caspase-3 assays.ResultsCa₃(PO₄)₂:Tb³⁺, Ce³⁺ phosphor exhibited needle like morphology with length and width ∼100-500 nm and ∼40-50 nm, respectively. It exhibited green emission at 550 nm corresponding to ⁵D₄→⁷F₅ transition with the CIE coordinates (x, y) as (0.284, 0.614). It also showed remarkable concentration dependent cytotoxicity against MCF-7 as well as MDA-MB 231 cells with negligible cytotoxicity compared to MCF-12A, a human epithelial healthy cell line. It reduced the proliferative index of both cell lines in a concentration dependent manner by inhibiting DNA synthesis and Ki67 protein. It also induced distinct apoptotic changes in the morphology of cell and nucleus and also activated the caspase-3 activity in breast cancer cell lines.ConclusionThe results suggest that Ca₃(PO₄)₂:Tb³⁺, Ce³⁺ phosphor may be useful for therapeutic application in clinical settings.     

Thermodynamics and molecular dynamics simulations of calcium binding to the regulatory site of human cardiac troponin C: evidence for communication with the structural calcium binding sites

Human cardiac troponin C (HcTnC), a member of the EF hand family of proteins, is a calcium sensor responsible for initiating contraction of the myocardium. Ca²⁺ binding to the regulatory domain induces a slight change in HcTnC conformation which modifies subsequent interactions in the troponin–tropomyosin–actin complex. Herein, we report a calorimetric study of Ca²⁺ binding to HcTnC. Isotherms obtained at 25 °C (10 mM 2-morpholinoethanesulfonic acid, 50 mM KCl, pH 7.0) provided thermodynamic parameters for Ca²⁺ binding to both the high-affinity and the low-affinity domain of HcTnC. Ca²⁺ binding to the N-domain was shown to be endothermic in 2-morpholinoethanesulfonic acid buffer and allowed us to extract the thermodynamics of Ca²⁺ binding to the regulatory domain. This pattern stems from changes that occur at the Ca²⁺ site rather than structural changes of the protein. Molecular dynamics simulations performed on apo and calcium-bound HcTnC_1–89 support this claim. The values of the Gibbs free energy for Ca²⁺ binding to the N-domain in the full-length protein and to the isolated domain (HcTnC_1–89) are similar; however, differences in the entropic and enthalpic contributions to the free energy provide supporting evidence for the cooperativity of the C-domain and the N-domain. Thermograms obtained at two additional temperatures (10 and 37 °C) revealed interesting trends in the enthalpies and entropies of binding for both thermodynamic events. This allowed the determination of the change in heat capacity (?C _p ) from a plot of ?H verses temperature and may provide evidence for positive cooperativity of Ca²⁺ binding to the C-domain.     

Cation binding sites on synaptic vesicles

The protein-sensitized fluorescence of Tb³⁺ was used as a probe for cation binding sites on synaptic vesicles. Competition studies show that the order of affinity for the sites is Cu²⁺ > Mn²⁺ > Ca²⁺ > Mg²⁺ and Zn²⁺ is inactive. Fluorescence quenching studies indicate that the site is superficial and the effect of pH suggests that histidine is involved in the binding. Measurements of enzyme activities in the presence of lanthanides reveal that the metal binding site identified by Tb³⁺ fluorescence is not the Cu²⁺ site associated with dopamine-β-hydroxylase. Terbium inhibits Ca²⁺-stimulated ATPase but not Mg²⁺-stimulated ATPase activities of the synaptic vesicle fraction. A kinetic analysis indicates that the site monitored by Tb³⁺ fluorescence may be a component of the Ca²⁺-stimulated ATPase. It is also suggested that Mg²⁺ and especially Cu²⁺ may bind to the sites in vivo, serving as a bridge between vesicles and other synaptic components such as the presynaptic plasma membrane.     

Thermodynamic linkage between calmodulin domains binding calcium and contiguous sites in the C-terminal tail of Ca(V)1.2

Calmodulin (CaM) binding to the intracellular C-terminal tail (CTT) of the cardiac L-type Ca²⁺ channel (Ca_V1.2) regulates Ca²⁺ entry by recognizing sites that contribute to negative feedback mechanisms for channel closing. CaM associates with Ca_V1.2 under low resting [Ca²⁺], but is poised to change conformation and position when intracellular [Ca²⁺] rises. CaM binding Ca²⁺, and the domains of CaM binding the CTT are linked thermodynamic functions. To better understand regulation, we determined the energetics of CaM domains binding to peptides representing pre-IQ sites A₁₅₈₈, and C₁₆₁₄ and the IQ motif studied as overlapping peptides IQ₁₆₄₄ and IQ′₁₆₅₀ as well as their effect on calcium binding. (Ca²⁺)₄-CaM bound to all four peptides very favorably (K_d ≤ 2 nM). Linkage analysis showed that IQ_1644-1670 bound with a K_d ~ 1 pM. In the pre-IQ region, (Ca²⁺)₂-N-domain bound preferentially to A₁₅₈₈, while (Ca²⁺)₂-C-domain preferred C₁₆₁₄. When bound to C₁₆₁₄, calcium binding in the N-domain affected the tertiary conformation of the C-domain. Based on the thermodynamics, we propose a structural mechanism for calcium-dependent conformational change in which the linker between CTT sites A and C buckles to form an A-C hairpin that is bridged by calcium-saturated CaM.     

Troponin Regulatory Function and Dynamics Revealed by H/D Exchange-Mass Spectrometry

Muscle contraction is tightly regulated by Ca²⁺ binding to the thin filament protein troponin. The mechanism of this regulation was investigated by detailed mapping of the dynamic properties of cardiac troponin using amide hydrogen exchange-mass spectrometry. Results were obtained in the presence of either saturation or non-saturation of the regulatory Ca²⁺ binding site in the NH₂ domain of subunit TnC. Troponin was found to be highly dynamic, with 60% of amides exchanging H for D within seconds of exposure to D₂O. In contrast, portions of the TnT-TnI coiled-coil exhibited high protection from exchange, despite 6 h in D₂O. The data indicate that the most stable portion of the trimeric troponin complex is the coiled-coil. Regulatory site Ca²⁺ binding altered dynamic properties (i.e. H/D exchange protection) locally, near the binding site and in the TnI switch helix that attaches to the Ca²⁺-saturated TnC NH₂ domain. More notably, Ca²⁺ also altered the dynamic properties of other parts of troponin: the TnI inhibitory peptide region that binds to actin, the TnT-TnI coiled-coil, and the TnC COOH domain that contains the regulatory Ca²⁺ sites in many invertebrate as opposed to vertebrate troponins. Mapping of these affected regions onto the troponin highly extended structure suggests that cardiac troponin switches between alternative sets of intramolecular interactions, similar to previous intermediate resolution x-ray data of skeletal muscle troponin.     

Luminescence and energy‐transfer properties of color‐tunable Ca2Mg0.25Al1.5Si1.25O7:Ce3+/Eu2+/Tb3+ phosphors for ultraviolet light‐emitting diodes

A series of Ca₂Mg_0.25Al_1.5Si_1.25O₇:Ce³⁺/Eu²⁺/Tb³⁺ phosphors was been prepared via a conventional high temperature solid‐state reaction and their luminescence properties were studied. The emission spectra of Ca₂Mg_0.25Al_1.5Si_1.25O₇:Ce³⁺,Eu²⁺ and Ca₂Mg_0.25Al_1.5Si_1.25O₇:Ce³⁺,Tb³⁺ phosphors show not only a band due to Ce³⁺ ions (409 nm) but also as a band due to Eu²⁺ (520 nm) and Tb³⁺ (542 nm) ions. More importantly, the effective energy transfer from Ce³⁺ to Eu²⁺ and Tb³⁺ ions was confirmed and investigated by emission/excitation spectra and luminescent decay behaviors. Furthermore, the energy level scheme and energy transfer mechanism were investigated and were demonstrated to be of resonant type via dipole–dipole (Ce³⁺ to Eu²⁺) and dipole–quadrupole (Ce³⁺ to Tb³⁺) reactions, respectively. Under excitation at 350 nm, the emitting color could be changed from blue to green by adjusting the relative doping concentration of Ce³⁺ and Eu²⁺ ions as well as Ce³⁺ and Tb³⁺ ions. The above results indicate that Ca₂Mg_0.25Al_1.5Si_1.25O₇:Ce³⁺,Eu²⁺/Tb³⁺ are promising single‐phase blue‐to‐green phosphors for application in phosphor conversion white‐light‐emitting diodes. Copyright © 2015 John Wiley & Sons, Ltd.     

Effects of Metal Ions on the Conformation and Activity of Acutolysin D from Agkistrodon Acutus Venom

Acutolysin D, isolated from the venom of Agkistrodon acutus, possesses marked haemorrhagic and proteolytic activities. The molecular weight and the absorption coefficients (A ^1% ₂₈₀) of acutolyisn D have been determined to be 47,850 ± 8 amu and 9.3 by mass spectrometer and UV spectrum, respectively. The effects of metal ions on the conformation and activity of acutolysin D have been studied by following fluorescence, circular dichroism and biological activity measurements. Acutolysin D contains two Ca²⁺-binding sites and two Zn²⁺-binding sites determined by atomic absorption spectrophotometer. Zn²⁺ is essential for the enzyme activities of acutolysin D, however, the presence of 1 mM Zn²⁺ significantly decreases its caseinolytic activity and intrinsic fluorescence intensity at pH 9.0 due to Zn(OH)₂ precipitate formation. Ca²⁺ is important for the structural integrity of acutolysin D, and the presence of 1 mM Ca²⁺ markedly enhances its caseinolytic activity. Interestingly, the caseinolytic activity which is inhibited partly by Cu²⁺, Co²⁺, Mn²⁺ or Tb³⁺ and inhibited completely by Cd²⁺, is enhanced by Mg²⁺. The fluorescence intensity of the protein decreases in the presence of Cu²⁺, Co²⁺, Cd²⁺ or Mn²⁺, but neither for Ca²⁺, Mg²⁺ nor for Tb³⁺. Zn²⁺, Ca²⁺, Mg²⁺, Cu²⁺, Mn²⁺, Co²⁺ and Tb³⁺ have slight effects on its secondary structure contents. In addition, Cd²⁺ causes a marked increase of antiparallel β-sheet content from 45.5% to 60.2%.     

Calcium binding to cardiac troponin C

The binding of Ca²⁺ to cardiac troponin C was studied by determining changes in the fluorescence and circular dichroism of the protein and by following changes in the free Ca²⁺ concentration by means of a Ca²⁺-specific electrode. Cardiac troponin C contains three Ca²⁺-binding sites which fall into two classes —two sites with a higher affinity and one with a lower affinity. The higher-affinity sites also bind Mg²⁺ which competes with the Ca²⁺.     

Fusion of human erythrocytes induced by uranyl acetate and rare earth metals

Incubation of human erythrocytes with either uranyl ions (UO₂²⁺) or rare earth metals (La³⁺, Nd³⁺, Sm³⁺, Eu³⁺, Tb³⁺, Dy³⁺ and Yb³⁺) at 37°C for 30–45 min resulted in the fusion of erythrocytes. Redistribution of membrane-associated particles was observed using colloidal-iron charge labelling and freeze-fracture electron microscopy. The fusion of erythrocytes induced by these agents, unlike Ca²⁺, did not exhibit the absolute requirement for phosphate. Moreover, agglutination and fusion by these agents was observed in neuraminidase-treated erythrocytes in contrast to Ca²⁺- and phosphate-induced fusion. Inhibitors of intrinsic transglutaminase activity partially inhibited (35–45%) the fusion induced by UO₂²⁺ suggesting that cross-linking of membrane proteins results in protein-free areas of lipid where fusion may be initiated.     

Terbium luminescence as a probe of the calcium binding site of trypsin and α-chymotrypsin

The large enhancement of the green luminescence of terbium ion which occurs on binding to porcine and bovine trypsins and to bovine α-chymotrypsin has been used to study the calcium binding sites of these enzymes. Excitation spectra, taken at low protein concentrations to minimize absorption effects, demonstrate that in each case, energy transfer occurs between the side chain of a tryptophan residue and bound Tb³⁺. Association constants for the binding of Tb³⁺ to the single binding site on each of the three proteins have been measured at 25 °C and pH 6.6. Ca²⁺ ions compete with Tb³⁺ for the single binding site, and association constants for Ca²⁺ were determined by Tb³⁺ displacement. The ratio of binding strengths of Ca²⁺ to α-chymotrypsin, bovine trypsin, porcine trypsin, and elastase is 1:12:24:23. Addition of Tb³⁺ to the homologous bacterial enzyme α-lytic protease caused no luminescence enhancement.     

Stable Structural Analog of Ca2+-ATPase ADP-insensitive Phosphoenzyme with Occluded Ca2+ Formed by Elongation of A-domain/M1′-linker and Beryllium Fluoride Binding

We have developed a stable analog for the ADP-insensitive phosphoenzyme intermediate with two occluded Ca²⁺ at the transport sites (E2PCa₂) of sarcoplasmic reticulum Ca²⁺-ATPase. This is normally a transient intermediate state during phosphoenzyme isomerization from the ADP-sensitive to ADP-insensitive form and Ca²⁺ deocclusion/release to the lumen; E1PCa₂ → E2PCa₂ → E2P + 2Ca²⁺. Stabilization was achieved by elongation of the Glu⁴⁰-Ser⁴⁸ loop linking the Actuator domain and M1 (1st transmembrane helix) with four glycine insertions at Gly⁴⁶/Lys⁴⁷ and by binding of beryllium fluoride (BeF_x) to the phosphorylation site of the Ca²⁺-bound ATPase (E1Ca₂). The complex E2Ca₂·BeF₃⁻ was also produced by lumenal Ca²⁺ binding to E2·BeF₃⁻ (E2P ground state analog) of the elongated linker mutant. The complex was stable for at least 1 week at 25 °C. Only BeF_x, but not AlF_x or MgF_x, produced the E2PCa₂ structural analog. Complex formation required binding of Mg²⁺, Mn²⁺, or Ca²⁺ at the catalytic Mg²⁺ site. Results reveal that the phosphorylation product E1PCa₂ and the E2P ground state (but not the transition states) become competent to produce the E2PCa₂ transient state during forward and reverse phosphoenzyme isomerization. Thus, isomerization and lumenal Ca²⁺ release processes are strictly coupled with the formation of the acylphosphate covalent bond at the catalytic site. Results also demonstrate the critical structural roles of the Glu⁴⁰-Ser⁴⁸ linker and of Mg²⁺ at the catalytic site in these processes.     

Effect of metals ions on thermostable alkaline phytase from Bacillus subtilis YCJS isolated from soybean rhizosphere soil

A Bacillus sp.YCJS strain showing phytase activity was isolated, and the phytase-encoding gene was cloned and expressed in Escherichia coli. The 1,149-bp full-length gene encoded a 26-residue putative signal peptide and a 356-residue mature protein. The molecular weight was estimated to be 47.5 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified recombinant enzyme phy(ycE) from E. coli exhibited a specific activity of 14 U mg^-1 protein. The optimum pH and temperature were 6.0 and 50 °C, respectively. The thermal stability of phy(ycE) was drastically improved in the presence of calcium ions (Ca²⁺). Fluorescence analysis results indicated that compared with phy(ycE) without added Ca²⁺, phy(ycE) in the presence of Ca²⁺ was more stable and the melting temperature improved from 47.8 to 62.4 °C. Circular dichroism spectrometric analysis revealed that the loss of enzymatic activity was most likely due to a conformational change, as the circular dichroism spectra of the holoenzyme and metal-depleted enzyme were significantly different. Compared with the Ca²⁺-reactivated enzyme, the La³⁺-reactivated enzyme did not undergo a significant recovery with respect to its conformation. The aromatic-sensitized terbium (Tb³⁺) fluorescence results indicated that five Tb³⁺ could bind to each molecule of phy(ycE) and that there were two high-affinity and three low-affinity binding sites.     

Ca2+-dependent Muscle Dysfunction Caused by Mutation of the Caenorhabditis elegans Troponin T-1 Gene

We have investigated the functions of troponin T (CeTnT-1) in Caenorhabditis elegans embryonic body wall muscle. TnT tethers troponin I (TnI) and troponin C (TnC) to the thin filament via tropomyosin (Tm), and TnT/Tm regulates the activation and inhibition of myosin-actin interaction in response to changes in intracellular [Ca²⁺]. Loss of CeTnT-1 function causes aberrant muscle trembling and tearing of muscle cells from their exoskeletal attachment sites (Myers, C.D., P.-Y. Goh, T. StC. Allen, E.A. Bucher, and T. Bogaert. 1996. J. Cell Biol. 132:1061–1077). We hypothesized that muscle tearing is a consequence of excessive force generation resulting from defective tethering of Tn complex proteins. Biochemical studies suggest that such defective tethering would result in either (a) Ca²⁺-independent activation, due to lack of Tn complex binding and consequent lack of inhibition, or (b) delayed reestablishment of TnI/TnC binding to the thin filament after Ca²⁺ activation and consequent abnormal duration of force. Analyses of animals doubly mutant for CeTnT-1 and for genes required for Ca²⁺ signaling support that CeTnT-1 phenotypes are dependent on Ca²⁺ signaling, thus supporting the second model and providing new in vivo evidence that full inhibition of thin filaments in low [Ca²⁺] does not require TnT.     

Fluorescence and circular dichroism studies with hemocyanin from taiwan snails

Several derivatives of hemocyanin from Taiwan snails (Achatina fulica) have been prepared. The reconstituted protein (R-HcO₂) has lower Cu content, lower circular dichroism intensity, and higher fluorescence intensity than native oxyhemocyanin (HcO₂). The Co(II) derivative (CoHc) does not take up molecular oxygen and only 50% of the total sites for Cu in native hemocyanin is taken up by Co. The half-apo derivative (half-apo-Hc) contains a single Cu per active site. Divalent cations quench the tryptophan fluorescence in the hemocyanin species and also quench the fluorescence from Tb³⁺ bound to the protein. The collisional quenching constants decrease in the order Co²⁺ > Mn²⁺ > Ca²⁺. The static component is negligible. For carboxy hemocyanin (HcCO), fluorescence originates from a Cu(I) CO complex and was used to study reaction of Hc CO with CN⁻.