Timolol binding to bovine ocular melanin in vitro.
The binding characteristics of 3H-timolol to bovine iris melanin were determined by using a ligand binding assay. The association and dissociation kinetics were performed using 100 nM 3H-timolol and 0.5 mg/ml melanin at 37 degrees C. The effects of pH, ethanol, 2.5 microM isoxuprine and 1 microM d- and 1-propranolol on 100 nM 3H-timolol binding were also determined. In saturation experiments 3H-timolol (from 1.25 nM to 5 microM) was equilibrated with 0.5 mg/ml melanin. The binding was saturable with the binding maximum of about 1 microM timolol/g melanin. The binding of 100 nM 3H-timolol to melanin increased up to 5 hours and amounted 24% of the added radioactivity. The best fit of the association constants was obtained by using a two fit model. The association rate constants were 4.92 x 10(5) M-1 min-1 and 5.95 x 10(6) M-1 min-1. The dissociation was rapid in vitro and uniphasic with a dissociation rate constant of 5.08 x 10(-3) min-1. The pH, ethanol and both enantiomers of propranolol did not appreciably alter the timolol binding, while isoxuprine diminished it.
Aula
P
P
Department of Pharmacology, University of Turku, Finland.
Kaila
T
T
Huupponen
R
R
Salminen
L
L
D000818:Animals; D001667:Binding, Competitive; D002417:Cattle; D000431:Ethanol; D005123:Eye / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007498:Iris; D007700:Kinetics; D008543:Melanins / Q000378:metabolism*; D011433:Propranolol / Q000494:pharmacology; D013237:Stereoisomerism; D013999:Timolol / Q000378:metabolism*
D008543:Melanins; D000431:Ethanol; D013999:Timolol; D011433:Propranolol