The affinity of human serum albumin for [3H]-digitoxin is dependent on albumin concentration.

Binding of [3H]-digitoxin to human serum albumin and human serum was investigated in order to characterize the relationship between binding and albumin concentration. Binding was determined by equilibrium dialysis at 37 degrees, 24 hr was required to reach equilibrium. Volume shift and protein dilution were avoided by adding dextran 70 to the buffer compartment. [3H]-Digitoxin binding both to purified albumin and to normal serum was markedly pH-dependent, the bound/unbound ratio being highly significantly (P < 0.001) inversely correlated to pH in the range 6-8.5. When albumin concentration was increased within the physiological range, the ratio bound/unbound [3H]-digitoxin increased much less than expected from predictions using the law of mass action. Binding saturation experiments revealed that the equilibrium dissociation constant for [3H]-digitoxin was increased at higher albumin concentrations without any decrease in the number of binding sites per albumin molecule. In conclusion, the results strongly indicate that binding estimates in therapeutic monitoring of digitoxin in patients with elevated or reduced albumin concentration should not be based on the law of mass action but on empiric relationships between albumin concentration and binding.

Ca2+ modulates the inhibition of (+)-[3H]isradipine binding by amiloride and quinacrine.

Binding studies were performed to characterize the inhibition by amiloride, 3,4-dichlorobenzamil and quinacrine of specific binding of (+)-[3H]isradipine to L-type voltage-operated calcium ion channels in rat cardiac membranes at 37 degrees C with and without 10(-3) M calcium added. By analysis of saturation, inhibition and dissociation curves we find that without the addition of calcium, amiloride (constant of inhibitor producing 50% inhibition (K0.5) = 6.9 x 10(-4) M, Hill coefficient (nH) = 1.99, k-1 increased) and 3,4-dichlorobenzamil (K0.5 = 7.7 x 10(-7) M, nH = 1.13, k-1 increased) inhibit (+)-[3H]isradipine binding by complex, allosteric interactions, suggesting positive cooperativity between sites for the inhibitors. Quinacrine (K0.5 = 6.7 x 10(-6) M, nH = 0.84, k-1 increased) inhibits the binding allosterically by an action compatible with binding to one site. Addition of 10(-3) M calcium affected the inhibition by amiloride (K0.5 = 1.02 x 10(-3) M, nH = 1.41) and quinacrine (K0.5 = 3.3 x 10(-5) M, nH = 0.65). With calcium added the mechanisms of inhibitions were complex, allosteric, and could be explained by positive cooperativity between sites for amiloride and negative cooperativity between sites for guinacrine. We conclude that calcium addition modulates the inhibitions by amiloride and quinacrine by increasing the inhibition constants and changing the cooperativity.

Inhibition by amiloride and quinacrine of specific [3H]nitrendipine binding to rat cardiac membranes.

Binding studies were designed to test if and how amiloride and quinacrine affected the specific binding of [3H]nitrendipine (NIT) to rat cardiac membranes. Specific binding of NIT was inhibited in a dose-dependent manner by amiloride [Hill coefficient (nH), 1.46; concentration of inhibitor producing 50% inhibition (K0.5) = 9.2 x 10(-4) M] and quinacrine (nH = 0.54, K0.5 = 7.7 x 10(-6) M). The inhibitions were incomplete in the presence of 10(-3) M Ca ions. The Hofstee plot was convex upwards for amiloride and concave upwards for quinacrine. Amiloride increased the Kd, decreased the maximum specific binding and increased the k-1. Quinacrine increased the Kd without changing the maximum specific binding and increased the k-1. The effects of amiloride and quinacrine on k-1 were nonadditive. We conclude that amiloride and quinacrine bind to or close to the L-type Ca channel, and inhibit the specific binding of NIT by allosteric, complex interactions influenced by the free concentration of Ca++. The nonadditive allosteric effects suggest a shared mechanism of interaction for amiloride and quinacrine with the site(s) of NIT. Several mechanisms are discussed to explain how amiloride and quinacrine can produce such inhibition of NIT binding.