Characterization of chicken cystatin binding to rat renal brush-border membranes

Influence of aminoglycoside antibiotics on chicken cystatin binding to renal brush-border membranes

“…The incubation of the constant amount of fluorescein‐labelled chicken cystatin with BBM suspension in the presence of increased concentrations of gentamicin revealed that gentamicin competed with chicken cystatin for the binding to BBM in a concentration‐dependent manner (Figure ). The calculated Ki (32 μ m ) for gentamicin, determining the affinity for the receptor, was several times higher than the equilibrium dissociation constant ( Kd = 3.62 μ m ) for chicken cystatin estimated in our previous study . Even the highest gentamicin concentration used in the experiment (100 μ m ) was not able to completely inhibit the chicken cystatin binding to BBM, showing that this antibiotic was not potent to significantly disrupt the interaction of cystatin with its receptor sites.…”

“…The equilibrium dissociation constant for gentamicin ( Ki ), determining the affinity of the receptor for the competitor, was calculated from the IC50 using the Cheng‐Prusoff equation: where IC50 denotes inhibitory concentration that causes 50% dissociation of bound labelled ligand and [ L ] is the concentration of FITC‐cystatin used in the experiment. Kd denotes the equilibrium dissociation constant of ligand and was established in our previous study …”

“…FITC-labelled cystatin binding to BBM in the presence of gentamicin was determined by the rapid filtration technique as described previously, and each experiment was carried out in three replicates. [15] In brief, 20 ml membrane suspension (100 mg protein per assay) was pre-incubated for 10 min at room temperature in 80 ml binding buffer (PBS, pH 7.4 with 0.02% Triton X-100). The reaction was initiated by the addition of 5 mm FITC-cystatin with increasing concentrations of gentamicin (0.5-100 mm) in incubation buffer.…”

“…Kd denotes the equilibrium dissociation constant of ligand and was established in our previous study. [15] Displacement studies…”

“…[14] Chicken cystatin and human cystatin C undergo the same endocytic pathway in the renal proximal tubular cells, which was established in independent studies. [15,16] The nature of chicken cystatin makes this protein a model toll for investigation of its human orthologue, therefore, we chose chicken cystatin in our study. To the best of our knowledge, we have quantified for the first time the competition between cystatin and aminoglycosides in an in-vitro model.…”