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Ca2+ Requirement (ca2+ + requirement)
Selected AbstractsMouse recombinant protein C variants with enhanced membrane affinity and hyper-anticoagulant activity in mouse plasmaFEBS JOURNAL, Issue 22 2009Michael J. Krisinger Mouse anticoagulant protein C (461 residues) shares 69% sequence identity with its human ortholog. Interspecies experiments suggest that there is an incompatibility between mouse and human protein C, such that human protein C does not function efficiently in mouse plasma, nor does mouse protein C function efficiently in human plasma. Previously, we described a series of human activated protein C (APC) Gla domain mutants (e.g. QGNSEDY-APC), with enhanced membrane affinity that also served as superior anticoagulants. To characterize these Gla mutants further in mouse models of diseases, the analogous mutations were now made in mouse protein C. In total, seven mutants (mutated at one or more of positions P10S12D23Q32N33) and wild-type protein C were expressed and purified to homogeneity. In a surface plasmon resonance-based membrane-binding assay, several high affinity protein C mutants were identified. In Ca2+ titration experiments, the high affinity variants had a significantly reduced (four-fold) Ca2+ requirement for half-maximum binding. In a tissue factor-initiated thrombin generation assay using mouse plasma, all mouse APC variants, including wild-type, could completely inhibit thrombin generation; however, one of the variants denoted mutant III (P10Q/S12N/D23S/Q32E/N33D) was found to be a 30- to 50-fold better anticoagulant compared to the wild-type protein. This mouse APC variant will be attractive to use in mouse models aiming to elucidate the in vivo effects of APC variants with enhanced anticoagulant activity. [source] Purification, crystallization and preliminary X-ray analysis of a ,-like calpainACTA CRYSTALLOGRAPHICA SECTION D, Issue 2 2003Gour P. Pal The X-ray structure of m-calpain in the absence of Ca2+ has been described, but it has not been possible to obtain sufficient ,-calpain for structure determination. Comparison of the two structures is of interest in attempting to understand their different Ca2+ requirements. Here, the crystallization in the absence of Ca2+ of an inactive mutant hybrid calpain (MW , 100,kDa), which contains 85% of the rat ,-calpain sequence and is well expressed in Escherichia coli, is described. The properties of this calpain in its active form and particularly its Ca2+ requirement are close to those expected for wild-type ,-calpain. Clusters of plate-shaped crystals were obtained by vapour diffusion with polyethylene glycol (Mr, 6000) as precipitating agent in the presence of detergent. The crystals diffract to a resolution of 2.7,Å at a synchrotron source. The space group is P21, with unit-cell parameters a = 72.7, b = 184.6, c = 86.3,Å, , = 100.7°. There are two molecules in the asymmetric unit, corresponding to a solvent content of 57.1%. [source] Chronic toxicity of lead to three freshwater invertebrates,Brachionus calyciflorus, Chironomus tentans, and Lymnaea stagnalisENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 1 2006Martin Grosell Abstract Chronic lead (Pb) toxicity tests with Brachionus calyciflorus, Chironomus tentans, and Lymnaea stagnalis were performed in artificial freshwaters. The no-observable-effect concentration (NOEC), lowest-observable-effect concentration (LOEC), and calculated 20% effect concentration (EC20) for the rotifer B. calyciflorus were 194, 284, and 125 ,g dissolved Pb/L, respectively. The midge C. tentans was less sensitive, with NOEC and LOEC of 109 and 497 ,g dissolved Pb/L, respectively, and the snail L. stagnalis exhibited extreme sensitivity, evident by NOEC, LOEC, and EC20 of 12, 16, and <4 ,g dissolved Pb/L, respectively. Our findings are presented in the context of other reports on chronic Pb toxicity in freshwater organisms. The L. stagnalis results are in agreement with a previous report on pulmonate snails and should be viewed in the context of current U.S. Environmental Protection Agency (U.S. EPA) hardness adjusted water quality criteria of 8 ,g Pb/L. The present findings and earlier reports indicate that freshwater pulmonate snails may not be protected by current regulatory standards. Measurements of whole-snail Na+ and Ca2+ concentrations following chronic Pb exposure revealed that Na+ homeostasis is disturbed by Pb exposure in juvenile snails in a complicated pattern, suggesting two physiological modes of action depending on the Pb exposure concentration. Substantially reduced growth in the snails that exhibit very high Ca2+ requirements may be related to reduced Ca2+ uptake and thereby reduced shell formation. [source] Purification, crystallization and preliminary X-ray analysis of a ,-like calpainACTA CRYSTALLOGRAPHICA SECTION D, Issue 2 2003Gour P. Pal The X-ray structure of m-calpain in the absence of Ca2+ has been described, but it has not been possible to obtain sufficient ,-calpain for structure determination. Comparison of the two structures is of interest in attempting to understand their different Ca2+ requirements. Here, the crystallization in the absence of Ca2+ of an inactive mutant hybrid calpain (MW , 100,kDa), which contains 85% of the rat ,-calpain sequence and is well expressed in Escherichia coli, is described. The properties of this calpain in its active form and particularly its Ca2+ requirement are close to those expected for wild-type ,-calpain. Clusters of plate-shaped crystals were obtained by vapour diffusion with polyethylene glycol (Mr, 6000) as precipitating agent in the presence of detergent. The crystals diffract to a resolution of 2.7,Å at a synchrotron source. The space group is P21, with unit-cell parameters a = 72.7, b = 184.6, c = 86.3,Å, , = 100.7°. There are two molecules in the asymmetric unit, corresponding to a solvent content of 57.1%. [source] | ||||||||||