Biomedical subjects

F Fernández-Belda

Publications and source records attributed to F Fernández-Belda.

3 recordsLinked to original sources

PubMed1987

Effect of the calcium-channel blockers on calcium accumulation in sarcoplasmic reticulum of skeletal muscle.

Vesicular fragments of sarcoplasmic reticulum isolated from rabbit skeletal muscle were actively loaded with Ca2+ in the presence of ATP and an ATP-regenerating system using Arsenazo III as metallochromic indicator to monitor Ca2+ movements across the membrane. Once the Ca2+ release is triggered by the presence of tetraphenylboron in the reaction medium, the addition of verapamil or diltiazem gives rise to a net Ca2+ entry inside the vesicles. Preincubation in the presence of verapamil does not abolish the tetraphenylboron-induced Ca2+ release, the verapamil-induced Ca2+ accumulation being still observed. There appears to be a high-affinity site for verapamil titrated in the micromolar concentration range, whereas diltiazem demonstrates more complex behavior when its concentration is raised. This study suggests the existence of a Ca2+ pathway (putative channels) which is blocked by the drugs tested allowing Ca2+ accumulation inside the vesicles owing to the Ca2+-dependent ATPase activity.

Adenosine Triphosphate↗

PubMed1986

A kinetic study of the irreversible inhibition of an enzyme measured in the presence of coupled enzymes. Fluorescein isothiocyanate as inhibitor of the adenosinetriphosphatase activity from sarcoplasmic reticulum.

A systematic procedure for the kinetic study of irreversible inhibition, when the enzymatic activity is measured in the presence of a coupled enzyme system, has been developed and analyzed. Simultaneous variation of the enzyme and inhibitor concentrations, maintaining a constant ratio between them, is recommended. The methodology is established to estimate the kinetic constants corresponding to the irreversible inhibitor. This approach is illustrated by the study of the inhibition of fluorescein isothiocyanate on the Ca2+-ATPase activity from sarcoplasmic reticulum measured in the presence of pyruvate kinase and lactate dehydrogenase as auxiliary enzymes. Treatment of the experimental data has been carried out by non-linear regression.

Animals↗

PubMed1985

Structural studies of mitochondrial coupling factor 1 using tyrosine fluorescence.

The absorbance and fluorescence spectral properties of mitochondrial F1-ATPase confirm that this protein does not contain tryptophan residues and therefore its fluorescence is due to tyrosines. The 36% increase in the fluorescence and the almost 100% increase in quantum yield upon denaturation of the protein suggest that a considerable number of tyrosyl residues have a very low quantum yield in the native enzyme. Quenching experiments using iodide indicate that all of the fluorophores are quenched and also all of them with the same quenching constant. These observations are interpreted as confirmatory of what has been found with several other proteins whose fluorescence originates from tyrosyl residues, where the buried tyrosines fluoresce with a much lower quantum yield than those which are exposed. ATP added to F1 previously depleted of loosely bound nucleotides changes the quenching constant of iodide and the quantum yield and this is interpreted to be due to a conformational change induced by the binding of the nucleotide to the enzyme. Addition of 2-mercaptoethanol decreases, although slightly, the polarization of the fluorescence. However, SDS addition gives a much bigger decrease. Hence disulphide bridges are less important for the tertiary structure of the protein than hydrophobic interactions, hydrogen bonding or other forces. Nevertheless the conformational change induced by reduction of disulphide bridges is detected in iodide quenching experiments and the change of the quantum yield of the enzyme.

Adenosine Triphosphate↗