Comparative properties of vertebrate parvalbumins.

Blum, Hubert E.; Lehky, Pavel; Kohler, Laure; Stein, Eric A.; Fischer, Edmond H.

doi:10.1016/s0021-9258(17)40438-8

Cited by 142 publications

(33 citation statements)

References 25 publications

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“…The contaminating Ca 2+ /Mg 2+ ions were removed from PA by precipitation of the protein by TCA [24], followed by gel-filtration according to [25] using Sephadex G-25 column equilibrated with a respective buffer.…”

Section: Removal Of Metal Ions From Pa Samplesmentioning

confidence: 99%

“…The measurements were performed in a CaF 2 cell with a pathlength of 4 µm in 10 mM H 3 BO 3 -KOH pH 9.0 buffer, either without metal ions or in the presence of 10 mM CaCl 2 /SrCl 2 . Apo-α-PA (metal-free form of α-PA) was prepared using combination of the TCA protein precipitation method [24] and Sephadex G-25 gel-filtration method [25]. Resolution was 2 cm −1 ; 256 spectra were averaged and the averaged spectrum of the buffer was subtracted.…”

Section: Ftir Spectroscopy Measurementsmentioning

confidence: 99%

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Strontium Binding to α-Parvalbumin, a Canonical Calcium-Binding Protein of the “EF-Hand” Family

et al. 2021

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Strontium salts are used for treatment of osteoporosis and bone cancer, but their impact on calcium-mediated physiological processes remains obscure. To explore Sr2+ interference with Ca2+ binding to proteins of the EF-hand family, we studied Sr2+/Ca2+ interaction with a canonical EF-hand protein, α-parvalbumin (α-PA). Evaluation of the equilibrium metal association constants for the active Ca2+ binding sites of recombinant human α-PA (‘CD’ and ‘EF’ sites) from fluorimetric titration experiments and isothermal titration calorimetry data gave 4 × 109 M−1 and 4 × 109 M−1 for Ca2+, and 2 × 107 M−1 and 2 × 106 M−1 for Sr2+. Inactivation of the EF site by homologous substitution of the Ca2+-coordinating Glu in position 12 of the EF-loop by Gln decreased Ca2+/Sr2+ affinity of the protein by an order of magnitude, whereas the analogous inactivation of the CD site induced much deeper suppression of the Ca2+/Sr2+ affinity. These results suggest that Sr2+ and Ca2+ bind to CD/EF sites of α-PA and the Ca2+/Sr2+ binding are sequential processes with the CD site being occupied first. Spectrofluorimetric Sr2+ titration of the Ca2+-loaded α-PA revealed presence of secondary Sr2+ binding site(s) with an apparent equilibrium association constant of 4 × 105 M−1. Fourier-transform infrared spectroscopy data evidence that Ca2+/Sr2+-loaded forms of α-PA exhibit similar states of their COO− groups. Near-UV circular dichroism (CD) data show that Ca2+/Sr2+ binding to α-PA induce similar changes in symmetry of microenvironment of its Phe residues. Far-UV CD experiments reveal that Ca2+/Sr2+ binding are accompanied by nearly identical changes in secondary structure of α-PA. Meanwhile, scanning calorimetry measurements show markedly lower Sr2+-induced increase in stability of tertiary structure of α-PA, compared to the Ca2+-induced effect. Theoretical modeling using Density Functional Theory computations with Polarizable Continuum Model calculations confirms that Ca2+-binding sites of α-PA are well protected against exchange of Ca2+ for Sr2+ regardless of coordination number of Sr2+, solvent exposure or rigidity of sites. The latter appears to be a key determinant of the Ca2+/Sr2+ selectivity. Overall, despite lowered affinity of α-PA to Sr2+, the latter competes with Ca2+ for the same EF-hands and induces similar structural rearrangements. The presence of a secondary Sr2+ binding site(s) could be a factor contributing to Sr2+ impact on the functional activity of proteins.

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Section: Removal Of Metal Ions From Pa Samplesmentioning

confidence: 99%

Section: Ftir Spectroscopy Measurementsmentioning

confidence: 99%

Strontium Binding to α-Parvalbumin, a Canonical Calcium-Binding Protein of the “EF-Hand” Family

et al. 2021

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“…The contaminating Ca 2+ /Mg 2+ ions were removed from rWT Ocm using precipitation by TCA [37] of the preliminary reduced protein (see above) (10-50 mM HEPES-KOH with/without 100 mM KCl, pH 7.0-8.2 or 10-20 mM H 3 BO 3 -KOH, pH 8.4-8.8 or 20 mM glycine-KOH, pH 9.2 or 10 mM Tris-HCl, pH 7.3-7.4 buffer), followed by purification from Ca 2+ /Mg 2+ and TCA by gel filtration method [38] using Sephadex G-25 column equilibrated with a respective buffer. To protect the reduced rWT Ocm from oxidation, 5 mM of freshly prepared DTT solution was added prior to the precipitation by TCA and before the gel filtration.…”

Section: Removal Of Metal Ions From Ocmmentioning

confidence: 99%

“…This procedure was used for metal removal from rWT/C18S Ocm for preparation of disulfide dimer of rWT Ocm and its redox studies, CD measurements of apo-and Mg 2+loaded Ocm, DSC measurements of Mg 2+ -loaded Ocm, chemical cross-linking and DLS measurements, bis-ANS fluorescence, and metal affinity studies. Only the gel filtration method [38] was used for spectrophotometric pH titrations of Ocm, CD measurements of Ca 2+ -loaded Ocm, DSC measurements of apo-and Ca 2+ -loaded Ocm, and kinetic studies of metal association/dissociation.…”

Section: Removal Of Metal Ions From Ocmmentioning

confidence: 99%

The Highly Conservative Cysteine of Oncomodulin as a Feasible Redox Sensor

et al. 2021

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Oncomodulin (Ocm), or parvalbumin β, is an 11–12 kDa Ca2+-binding protein found inside and outside of vertebrate cells, which regulates numerous processes via poorly understood mechanisms. Ocm consists of two active Ca2+-specific domains of the EF-hand type (“helix-loop-helix” motif), covered by an EF-hand domain with inactive EF-hand loop, which contains a highly conservative cysteine with unknown function. In this study, we have explored peculiarities of the microenvironment of the conservative Cys18 of recombinant rat Ocm (rWT Ocm), redox properties of this residue, and structural/functional sensitivity of rWT Ocm to the homologous C18S substitution. We have found that pKa of the Cys18 thiol lays beyond the physiological pH range. The measurement of redox dependence of rWT Ocm thiol–disulfide equilibrium (glutathione redox pair) showed that redox potential of Cys18 for the metal-free and Ca2+-loaded protein is of −168 mV and −176 mV, respectively. Therefore, the conservative thiol of rWT Ocm is prone to disulfide dimerization under physiological redox conditions. The C18S substitution drastically reduces α-helices content of the metal-free and Mg2+-bound Ocm, increases solvent accessibility of its hydrophobic residues, eliminates the cooperative thermal transition in the apo-protein, suppresses Ca2+/Mg2+ affinity of the EF site, and accelerates Ca2+ dissociation from Ocm. The distinct structural and functional consequences of the minor structural modification of Cys18 indicate its possible redox sensory function. Since some other EF-hand proteins also contain a conservative redox-sensitive cysteine located in an inactive EF-hand loop, it is reasonable to suggest that in the course of evolution, some of the EF-hands attained redox sensitivity at the expense of the loss of their Ca2+ affinity.

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“…Bu nedenle hızlı kasılan kaslarda PA düzeyleri yüksektir. Denemeler; dekalsifiye olmuş PA 'lerin, miyofibrillerin ATP-az aktivitesini tamamen bloke ettiğini, sarkoplazmik retikulumun PA'leri kalsifiye etmeğe yetenekli olduğunu göstermiştir (2,4,10). Kaslar total PA miktarı ve izotip yönünden farklılıklar gösterir.…”

Section: Arif Altıntaş2unclassified

TURNA BALı Gl BEYAZ KASLARıNDAN PARVALBUMtNLERİN SEPARASYONU VE SAFLAŞTIRILMASI'

Altintaş¹

1986

Ankara Üniversitesi Veteriner Fakültesi Dergisi

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Comparative properties of vertebrate parvalbumins.

Cited by 142 publications

References 25 publications

Strontium Binding to α-Parvalbumin, a Canonical Calcium-Binding Protein of the “EF-Hand” Family

Strontium Binding to α-Parvalbumin, a Canonical Calcium-Binding Protein of the “EF-Hand” Family

The Highly Conservative Cysteine of Oncomodulin as a Feasible Redox Sensor

TURNA BALı Gl BEYAZ KASLARıNDAN PARVALBUMtNLERİN SEPARASYONU VE SAFLAŞTIRILMASI'

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