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Notes: The binding of lipophilic radioligands to homogenized tissue was investigated with the help of a simple, two-component model: a specific component reflects binding to a single and uniform population of sites; a nonspecific component reflects partitioning into the membrane and the entrapment of some drug present in the aqueous phase prior to separation of the pariculate fraction. The results indicate that the capacity and the affinity of the receptor may be underestimated when the data are analyzed in terms of total rather than free radioligand. Errors in capacity arise when for a significant fraction of the radioligand access to the receptor is blocked by an unlabelled drug and this appears as nonspecific binding. This is most likely to occur when the partition coefficient is such that the free radioligand is located pre-dominantly in the particulate phase. Errors in affinity reflect the tendency of the membrane to reduce the free concentration of a lipophilic drug in the aqueous phase. A further complication arises when a significant fraction of the total radioligand binds to the receptor. [3H]Spiperone binds to dopamine D2 receptors with a dissociation constant of about 50 pM and partitions into the particulate phase of brain homogenates with a membrane/buffer partition coefficient of 410. As expected, both capacity and affinity can appear to depend on the concentration of tissue used in the assay. If the partition coefficient is known, corrected estimates of both parameters can be obtained knowing only the total concentration of radioligand; if the partition coefficient is not known, the free concentration of radioligand in the aqueous phase must be measured independently. The former procedure requires that the aqueous and particulate components of the system be separated by centrifugation; with filtration, the removal of an indeterminate amount of radioligand from the membrane during washing precludes any correction based on the partition coefficient. For the specific example of [3H]spiperone in human brain, the artifacts become negligible at concentrations of protein below 0.1 mg/ml of incubate. The capacity per unit of original tissue is best determined using unwashed preparations, since about 30% of the total protein and a comparable percentage of the receptors are lost on washing.