Characterization of a region of the primary sequence of troponin C involved in calcium ion-dependent interaction with troponin I.
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ABSTRACT:
SUBMITTER: Weeks RA
PROVIDER: S-EPMC1185798 | biostudies-other | 1978 Aug
REPOSITORIES: biostudies-other
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Project description:Calcium sensitizers enhance the transduction of the Ca(2+) signal into force within the heart and have found use in treating heart failure. However the mechanisms of action for most Ca(2+) sensitizers remain unclear. To address this issue an efficient fluorescence based approach to Ca(2+) sensitizer screening was developed which monitors cardiac troponin C's (cTnC's) hydrophobic cleft. This approach was tested on four common Ca(2+) -sensitizers, EMD 57033, levosimendan, bepridil and pimobendan with the aim of elucidating the mechanisms of action for each as well as proving the efficacy of the new screening method. Ca(2+) -titration experiments were employed to determine the effect on Ca(2+) sensitivity and cooperativity of cTnC opening, while stopped flow experiments were used to investigate the impact on cTnC relaxation kinetics. Bepridil was shown to increase the sensitivity of cTnC for Ca(2+) under all reconstitution conditions, sensitization by the other drugs was context dependent. Levosimendan and pimobendan reduced the rate of cTnC closing consistent with a stabilization of cTnC's open conformation while bepridil increased the rate of relaxation. Experiments were also run on samples containing cTnT(T204E), a known Ca(2+) -desensitizing phosphorylation mimic. Levosimendan, bepridil, and pimobendan were found to elevate the Ca(2+) -sensitivity of cTnT(T204E) containing samples in this context.
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Project description:Calcium-dependent protein kinases (CDPKs) are considered promising targets for pharmaceutical intervention of cryptosporidiosis. Whole-genome sequencing has revealed the presence of several CDPKs (CpCDPKs) in Cryptosporidium parvum. In this study, we expressed recombinant CpCDPK3 encoded by the cgd5_820 gene in Escherichia coli. The biologic characteristics and functions of CpCDPK3 were examined using qRT-PCR, immunofluorescence microscopy, and in vitro neutralization assay. The expression of the cgd5_820 gene peaked in merozoites during in vitro culture while the CpCDPK3 protein was expressed in both sporozoites and merozoites. Polyclonal antibodies against CpCDPK3 showed no significant inhibitory effects on host invasion by the parasites. We assessed the inhibitory effects of 46 candidate compounds from molecular docking of CpCDPK3 on both C. parvum development and CpCDPK3 enzyme activities. One compound was identified to be effective. Results of these analyses suggest that CpCDPK3 might play an important role in the growth of C. parvum.
| S-EPMC7225609 | biostudies-literature
Project description:1. Eight peptides were separated from the CNBr digest of troponin T from rabbit white skeletal muscle and characterized. 2. By study of the amino acid sequence of the methionine-containing peptides isolated after chymotryptic and tryptic digestion and of the N- and C-terminals of the CNBr peptides, six of the latter were shown to be arranged in the sequence CNB1-CNB2-CNB5-CNB6-CNB8-CNB7. The other two peptides, CNB1' and CNB3, have been shown to be partial digestion products. 3. The CNBr peptides CNB1' and CNB2 contained a common sequence and were the only peptides in CNBr digests of troponin T that formed a complex with tropomyosin as judged by viscometric and electrophoretic studies. 4. It is concluded that tropomyosin interacts with the N-terminal half of the troponin T molecule approximately in the region lying between residues 70 and 160. 5. Electrophoretic evidence indicates that tropomyosin and troponin C interact with troponin T. 6. None of the major CNBr peptides of troponin T isolated formed a complex with troponin C on electrophoresis at pH 8.6.
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Project description:1. The molecular weight of the calcium-binding protein of rabbit white skeletal muscle was estimated to be 18500 by sedimentation equilibrium and electrophoresis in sodium dodecyl sulphate. 2. Addition of 2 Ca(2+) ions per molecule produced reversible changes in the u.v.-absorption spectrum that are interpreted as arising from conformational changes in the structure of the protein. 3. Cd(2+) was almost as effective as Ca(2+) in producing the spectral changes. Other bivalent metal ions, particularly Mg(2+), were less effective. 4. Binding of Ca(2+) by the calcium-binding protein produced an increase in mobility to the anode on electrophoresis in 6m-urea at pH8.6. The Ca(2+)-saturated form of the protein was more retarded on gel filtration than the Ca(2+)-free form. 5. In the presence of Ca(2+) the calcium-binding protein formed an equimolar complex with the inhibitory protein. This complex was stable in 8m-urea and in the pH range 7.0-8.6. 6. An isotope-dilution method for the measurement of the content of calcium-binding protein in whole muscle is described. In rabbit psoas muscle the ratio of actin monomers to molecules of calcium-binding protein was approx. 7:1. Similar values were obtained for red skeletal and cardiac muscle. 7. Evidence is presented indicating that in the rabbit the inhibitory protein of the troponin complex of red skeletal and cardiac muscles is different from the inhibitory protein of white skeletal muscle.
| S-EPMC1166100 | biostudies-other
Project description:In the presence of ATP and of Mg(2+), human erythrocyte membranes show a phosphatase activity towards p-nitrophenyl phosphate which is activated by low concentrations of Ca(2+). The effect of Ca(2+) is strongly enhanced if either K(+) or Na(+) is also present. Activation of the p-nitrophenyl phosphate phosphatase by Ca(2+) reaches a half-maximum at about 8mum-Ca(2+) and is apparent only when the ion has access to the inner surface of the cell membrane. Ca(2+)-dependent phosphatase activity can only be observed if ATP is at the inner surface of the cell membrane, and the presence of ATP seems to be absolutely necessary, since either its removal or its replacement by other nucleoside triphosphates abolishes the activating effect of Ca(2+). The properties of the (ATP+Ca(2+))-dependent phosphatase are very similar to those of the Ca(2+)-dependent ATPase (adenosine triphosphatase), also present in erythrocyte membranes, which probably is involved in Ca(2+) transport in erythrocytes. The similarities suggest that both activities may be properties of the same molecular system. This view is further supported by the fact that p-nitrophenyl phosphate inhibits to a similar extent Ca(2+)-dependent ATPase activity and ATP-dependent Ca(2+) extrusion from erythrocytes.
| S-EPMC1165937 | biostudies-other
Project description:Microsomal fraction was prepared by ultracentrifugation of homogenates of cortical tissue from bovine brains. The preparation displayed ATPase (adenosine triphosphatase) activity in the presence of Mg(2+) (6.4mumol of P(i)/h per mg of protein) and Ca(2+) (3.4mumol of P(i)/h per mg of protein). Kinetic analysis of the activation of the enzyme preparation by Ca(2+) resulted in the demonstration of two apparent K(m) values for Ca(2+) (6.0x10(-8)m and 1.2x10(-6)m). Treatment of the microsomal membranes with Triton X-100 resulted in solubilization of the ATPase, though with some loss of activity. The solubilized microsomal proteins were incorporated into liposomes. By incubation of the liposomes in media containing (45)Ca(2+) an ATP-dependent uptake of Ca(2+) was demonstrated. The solubilized preparation was subjected to preparative isoelectric focusing in granulated gel beds. Two distinct peaks of Mg(2+)- and Ca(2+)-dependent ATPase activity were observed at pH4.8 (peak 4.8) and at pH6.3 (peak 6.3). The material isolated in peaks 4.8 and 6.3 was focused in polyacrylamide gel with pH gradients. The material corresponding to peak 4.8 consisted of a single protein, whereas peak 6.3 contained one major and at least one minor protein. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis confirmed these results and indicated that the major component of peak 4.8 and the protein of peak 6.3 both had a molecular weight of 105000. The material in peaks 4.8 and 6.3 was assayed for ATPase activity in the presence of various concentrations of Ca(2+). Kinetic analysis of the results for peak 4.8 demonstrated an apparent K(m) value for Ca(2+) of 4.1x10(-8)m. The enzyme isolated at pH6.3 had an apparent K(m) value of 3.8x10(-6)m. However, when the material from peak 4.8 was incubated in the presence of 1mm-Mg(2+) the ATPase could not be activated by Ca(2+).
| S-EPMC1161164 | biostudies-other
Project description:The microtubule cytoskeleton supports cellular morphogenesis and polar growth, but the underlying mechanisms are not understood. In a screen for morphology mutants defective in microtubule organization in the fungus Ustilago maydis, we identified eca1 that encodes a sarcoplasmic/endoplasmic calcium ATPase. Eca1 resides in the endoplasmic reticulum and restores growth of a yeast mutant defective in calcium homeostasis. Deletion of eca1 resulted in elevated cytosolic calcium levels and a severe growth and morphology defect. While F-actin and myosin V distribution is unaffected, Deltaeca1 mutants contain longer and disorganized microtubules that show increased rescue and reduced catastrophe frequencies. Morphology can be restored by inhibition of Ca(2+)/calmodulin-dependent kinases or destabilizing microtubules, indicating that calcium-dependent alterations in dynamic instability are a major cause of the growth defect. Interestingly, dynein mutants show virtually identical changes in microtubule dynamics and dynein-dependent ER motility was drastically decreased in Deltaeca1. This indicates a connection between calcium signaling, dynein, and microtubule organization in morphogenesis of U. maydis.
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Project description:Cytoplasmic calcium (Ca2+) activates the bestrophin anion channel, allowing chloride ions to flow down their electrochemical gradient. Mutations in bestrophin 1 (BEST1) cause macular degenerative disorders. Previously, we determined an X-ray structure of chicken BEST1 that revealed the architecture of the channel. Here, we present electrophysiological studies of purified wild-type and mutant BEST1 channels and an X-ray structure of a Ca2+-independent mutant. From these experiments, we identify regions of BEST1 responsible for Ca2+ activation and ion selectivity. A "Ca2+ clasp" within the channel's intracellular region acts as a sensor of cytoplasmic Ca2+. Alanine substitutions within a hydrophobic "neck" of the pore, which widen it, cause the channel to be constitutively active, irrespective of Ca2+. We conclude that the primary function of the neck is as a "gate" that controls chloride permeation in a Ca2+-dependent manner. In contrast to what others have proposed, we find that the neck is not a major contributor to the channel's ion selectivity. We find that mutation of a cytosolic "aperture" of the pore does not perturb the Ca2+ dependence of the channel or its preference for anions over cations, but its mutation dramatically alters relative permeabilities among anions. The data suggest that the aperture functions as a size-selective filter that permits the passage of small entities such as partially dehydrated chloride ions while excluding larger molecules such as amino acids. Thus, unlike ion channels that have a single "selectivity filter," in bestrophin, distinct regions of the pore govern anion-vs.-cation selectivity and the relative permeabilities among anions.
| S-EPMC5127342 | biostudies-literature
Project description:TMEM16A, a Ca2+-sensitive Cl- channel, plays key roles in many physiological functions related to Cl- transport across lipid membranes. Activation of this channel is mediated via binding intracellular Ca2+ to the channel with a relatively high apparent affinity, roughly in the sub-μM to low μM concentration range. Recently available high-resolution structures of TMEM16 molecules reveal that the high-affinity Ca2+ activation sites are formed by several acidic amino acids, using their negatively charged sidechain carboxylates to coordinate the bound Ca2+. In this study, we examine the interaction of TMEM16A with a divalent cation, Co2+, which by itself cannot activate current in TMEM16A. This divalent cation, however, has two effects when applied intracellularly. It inhibits the Ca2+-induced TMEM16A current by competing with Ca2+ for the aforementioned high-affinity activation sites. In addition, Co2+ also potentiates the Ca2+-induced current with a low affinity. This potentiation effect requires high concentration (mM) of Co2+, similar to our previous findings that high concentrations (mM) of intracellular Ca2+ ([Ca2+]i) can induce more TMEM16A current after the Ca2+-activation sites are saturated by tens of μM [Ca2+]i. The degrees of potentiation by Co2+ and Ca2+ also roughly correlate with each other. Interestingly, mutating a pore residue of TMEM16A, Y589, alters the degree of potentiation in that the smaller the sidechain of the replaced residue, the larger the potentiation induced by divalent cations. We suggest that the Co2+ potentiation and the Ca2+ potentiation share a similar mechanism by increasing Cl- flux through the channel pore, perhaps due to an increase of positive pore potential after the binding of divalent cations to phospholipids in the pore. A smaller sidechain of a pore residue may allow the pore to accommodate more phospholipids, thus enhancing the current potentiation caused by high concentrations of divalent cations.
| S-EPMC7164836 | biostudies-literature
Project description:Ca2+ regulation of arthropod actomyosin adenosine triphosphatase is associated with both the thin filaments, as in vertebrates, and with the myosin, as in molluscs. The actomyosin of decapod-crustacean fast muscles was previously considered to be an exception, displaying only a Ca2+-regulatory system linked to the thin filaments and not a myosin-linked regulatory system. In the present study, myosin regulation is demonstrated in a variety of decapod muscles when they are tested under more physiological ionic conditions. Myosin regulation is shown by using mixtures of pure rabbit actin with myofibrils, with actomyosin and with purified myosin, and in each case the adenosine triphosphatase is Ca2+ dependent. Myosin regulation may also occur in vertebrate striated muscle, but seemingly is lost during purification of the myosin.
| S-EPMC1164695 | biostudies-other