Binding of bumetanide to microsomes from optic ganglia of the squid, Loligo pealei.
Saturable high-affinity binding of [3H] bumetanide [dissociation constant (KD) = 80 nM] was measured in microsomal membranes prepared from squid optic ganglia. Under control conditions, the maximal specific binding of labeled bumetanide (Bmax) was approximately 6-7 pmol/mg protein. Binding had a higher relative affinity for bumetanide than for furosemide and depended on the presence of Cl- and K+, but not Na+, in the incubation media. In the case of K+, [3H]bumetanide binding was half-saturated at [K+] = 100 mM. The Cl- effect was biphasic. At [Cl-] between 0 and 150 mM, [3H]bumetanide binding increased with increasing [Cl-]. However, when [Cl-] was increased above 150 mM, [3H]bumetanide binding was progressively reduced. ATP acted as a nonessential activator [mean affinity constant (K0.5) approximately 1 microM] of the ion-dependent [3H]bumetanide binding by increasing the apparent binding capacity. The activation by ATP did not require Mg2+. Other adenosine analogues also stimulated the binding of bumetanide.
Altamirano
A A
AA
Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston 77550.
Watts
B A
BA
Russell
J M
JM
D000227:Adenine Nucleotides / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000378:metabolism*; D002034:Bumetanide / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism; D002794:Choline / Q000494:pharmacology; D049832:Decapodiformes; D004232:Diuretics / Q000378:metabolism*; D005724:Ganglia / Q000378:metabolism*; D007425:Intracellular Membranes / Q000187:drug effects / Q000378:metabolism; D007700:Kinetics; D008433:Mathematics; D008861:Microsomes / Q000378:metabolism*; D008954:Models, Biological; D011188:Potassium / Q000378:metabolism / Q000494:pharmacology; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology
D000227:Adenine Nucleotides; D002712:Chlorides; D004232:Diuretics; D000644:Quaternary Ammonium Compounds; D002034:Bumetanide; D000255:Adenosine Triphosphate; C027917:tetramethylammonium; D002794:Choline; D011188:Potassium